In Vitro Cell.Dev.Biol.—Plant (2011) 47:148–156
DOI 10.1007/s11627-010-9316-5
INVITED REVIEW
Comparative in vitro germination ecology of Calopogon
tuberosus var. tuberosus (Orchidaceae) across
its geographic range
Philip J. Kauth & Michael E. Kane &
Wagner A. Vendrame
Received: 9 November 2009 / Accepted: 28 September 2010 / Published online: 26 October 2010 / Editor: P. Lakshmanan
# The Society for In Vitro Biology 2010
Abstract Seed responses to temperature are often essential
to the study of germination ecology, but the ecological role
of temperature in orchid seed germination remains uncertain. The response of orchid seeds to cold stratification have
been studied, but the exact physiological role remains
unclear. No studies exist that compare the effects of either
cold stratification or temperature on germination among
distant populations of the same species. In two separate
experiments, the role of temperature (25, 22/11, 27/15, 29/
19, 33/24°C) and chilling at 10°C on in vitro seed
germination were investigated using distant populations of
Calopogon tuberosus var. tuberosus. Cooler temperatures
promoted germination of Michigan seeds; warmer temperatures promoted germination of South Carolina and north
central Florida seeds. South Florida seed germination was
highest under both warm and cool temperatures. More
advanced seedling development generally occurred at
higher temperatures with the exception of south Florida
seedlings, in which the warmest temperature suppressed
development. Fluctuating diurnal temperatures were more
beneficial for germination compared to constant temperatures. Cold stratification had a positive effect on germination among all populations, but South Carolina seeds
required the longest chilling treatments to obtain maximum
germination. Results from the cold stratification experiment
P. J. Kauth (*) : M. E. Kane
Plant Restoration, Conservation and Propagation Biotechnology
Program, Environmental Horticulture Department,
University of Florida,
PO Box 110675, Gainesville, FL 32611, USA
e-mail: pkauth@ufl.edu
W. A. Vendrame
Tropical Research and Education Center, University of Florida,
18905 SW 280th,
St Homestead, FL 33031, USA
indicate that a physiological dormancy is present, but the
degree of dormancy varies across the species range. The
variable responses among populations may indicate ecotypic
differentiation.
Keywords Cold stratification . Diurnal temperatures .
Ecotype . In vitro ecology . Physiological dormancy . Orchid
Introduction
Understanding germination ecology is essential to further
our knowledge about the timing of germination, seed
maturation, seed dispersal, dormancy, and environmental
cues that promote germination (Baskin and Baskin 2001;
Donohue 2005). While examining in situ germination is
ideal, studying the germination ecology of orchid seeds in
situ is difficult and time consuming because orchid seeds
are minute and germination is often low (Brundrett et al.
2003; Zettler et al. 2005; Diez 2007; Øien et al. 2008).
Alternative to in situ germination, asymbiotic in vitro
techniques have been developed to successfully germinate
orchid seeds (Kauth et al. 2008a). Ex situ conditions,
including in vitro studies, are often used to study
germination ecology; however, these conditions provide
an artificial environment not encountered in situ. Ecological
data can be carefully extrapolated from ex situ studies by
controlling factors, such as photoperiod and temperature to
interpret field germination (Donohue 2005). In addition,
studying seed germination ecology under ex situ and in vitro
conditions is also an effective method to detect ecotypic
differentiation in widespread populations (Seneca 1972;
Singh 1973; Seneca 1974; Probert et al. 1985). Using in
vitro ecology techniques can be an effective method to study
ecotypic differentiation (Kauth et al. 2008b). Studying
COMPARATIVE IN VITRO GERMINATION ECOLOGY OF CALOPOGON TUBEROSUS
ecotypic differentiation is necessary for restoration programs
since introducing mal-adapted ecotypes may reduce plant
population fitness (Hufford and Mazer 2003; McKay et al.
2005). However, intraspecific seed germination of orchid
species has received little attention.
For many plant species, temperature is a major factor
responsible for the onset and breaking of seed dormancy
(Baskin and Baskin 2004). Although temperature has been
previously studied on orchid seeds (Kauth et al. 2008a), its
exact ecological role remains uncertain, especially in populations of the same species. For this reason, using non-orchid
seed studies as framework to understand temperature effects
on orchid seed germination is necessary. The role of constant
temperatures has been examined in orchid seed germination
(Harvais 1973; Rasmussen et al. 1990; Rasmussen and
Rasmussen 1991; Rasmussen 1992; Mweetwa et al. 2008),
but using constant temperatures may not reflect true environmental conditions (Baskin et al. 2006). However, constant
temperatures improved in vitro germination of several orchids
over alternating temperatures (van Waes and Debergh 1986).
Regardless, selecting temperatures similar to those found in
situ and replicating them under controlled conditions may lead
to better insight into germination ecology.
Likewise, cold stratification of orchid seeds has been
previously examined, but its physiological role in orchid seed
germination is not well-understood. However, cold stratification has been reported to increase germination of many
terrestrial orchid species (van Waes and Debergh 1986;
Rasmussen 1992; De Pauw and Remphrey 1993; Chu and
Mudge 1994; Tomita and Tomita 1997; Miyoshi and Mii
1998; Zettler et al. 2001; Shimura and Koda 2004, 2005;
Øien et al. 2008). In non-orchids, cold stratification has been
reported to decrease enzymatic reactions, slow metabolic
processes that inhibit germination and change enzyme
production and concentrations (Bewley and Black 1994).
The effects of cold stratification among distant populations
of the same orchid species have not been reported.
Calopogon tuberosus var. tuberosus (L.) Britton, Sterns,
Poggenberg is a terrestrial orchid native to eastern North
America. C. tuberosus occupies diverse habitats such as marl
prairies, pine flatwoods, roadsides, fens, and sphagnum bogs
(Luer 1972). Morphological and genetic variation exist
throughout the range of C. tuberosus, but ecotypes have
not been defined (Goldman et al. 2004a, b; Trapnell et al.
2004). Variation in biomass allocation, timing of corm
formation, and seed germination responses to photoperiod
and germination media provided insight into possible
ecotypic differentiation in C. tuberosus (Kauth et al.
2008b; Kauth and Kane 2009). However, the effects of
temperature on seed germination were not previously
studied. Our present study differs by examining temperature
and cold stratification effects on the in vitro germination
ecology of widespread C. tuberosus populations. Since C.
149
tuberosus populations are found in areas with different
maximum and minimum temperatures (Fig. 1), this may be
a selection pressure influencing germination ecology.
Two separate experiments were conducted to examine
the role of temperature and cold-stratification on the in vitro
asymbiotic germination among widespread C. tuberosus
populations. Our hypotheses were as follows: (1) higher
germination percentages will occur under diurnal rather
than constant temperatures. (2) Seeds from northern
populations will require lower temperatures to germinate.
(3) Chilling seeds will increase overall germination due to
relieving dormancy. (4) Seeds from northern populations
will require longer chilling periods.
Materials and Methods
Seed sources and collection. For each population, light
green to yellowing intact seed capsules were collected
before dehiscence throughout summer 2007 from at least
three mother plants (three capsules each). Capsules were
collected from south Florida (Collier County, FL), north
central Florida (Levy County, FL), three locations in
South Carolina (Greenville County, South Carolina), and
the northern Michigan peninsula (Menominee County,
Michigan; Table 1). Intact capsules were collected to
reduce potential surface contamination of individual
seeds. Following collection and receipt, seed capsules
were pooled by population and stored at 23°C over silica
desiccant for 2 wk. After 2 wk, seeds were removed from
the capsules, dried over silica desiccant, and stored at
−11°C until use.
Embryo viability testing (Table 1) was modified according
to Kauth et al. (2008b) using 2, 3, 5 triphenyl tetrazolium
chloride (TTC). Testing was performed on mature seeds after
capsules dehisced at room temperature over desiccant. Seeds
were scarified in an aqueous 5% CaOCl2 solution for 3 h
before staining in TTC (Kauth et al. 2008b). Two replications of 100 seeds each were used for each population
(200 seeds total). Embryos with any degree of red
pigmentation were considered viable.
Medium and seed preparation for all experiments. BM-1
Terrestrial Orchid germination medium (B141; PhytoTechnology Laboratories, Shawnee Mission, KS) was selected based
on prior germination success with C. tuberosus seed sources
(Kauth et al. 2008b). The medium was supplemented with
0.1% activated charcoal. Medium pH was adjusted to 5.7 with
0.1N KOH prior to autoclaving for 40 min at 117.7 kPa and
121°C. Forty milliliter sterile medium was dispensed into
square 100×15 mm Petri plates with a 36-cell bottom
(Integrid™ Petri Dish, Becton Dickinson and Company,
Woburn, MA).
150
KAUTH ET AL.
Figure 1. Monthly temperatures
at each location where seed collections occurred in the present
study. Average temperatures are
the average of daily lows and
highs over an entire month. Data
from National Weather Service
(http://www.weather.gov/) was
last retrieved on February 18,
2009. Temperature data were
obtained from the closest city
within 50 km to specific sites.
Michigan: Escanaba, MI; South
Carolina: Greenville, SC;
North Central Florida: Ocala,
FL; South Florida:
Naples, FL.
Mature seeds were surface sterilized in sterile scintillation
vials for 3 min in a solution of 5 mL absolute ethanol, 5 mL
6% NaOCl and 90 mL sterile distilled–deionized (dd) water.
Seeds were rinsed twice with sterile dd water after surface
sterilization. Seeds were then placed on the surface of the
germination media with a 10 μl sterile inoculating loop. The
interior 16 cells of the Petri plates were used for subreplications to avoid uneven media drying at the edges. Three of the
interior 16 cells were selected randomly for subreplication.
Petri plates were sealed with one layer of Nescofilm (Karlan
Research Products, Santa Rosa, CA).
Temperature effects on seed germination. Fluctuating diurnal temperatures were selected based on average seasonal d and
night temperatures in Gainesville, FL: 33/24°C (summer); 29/
19°C (spring); 27/15°C (fall); 22/11°C (winter). A constant
temperature of 25°C was also used. Five replications each were
used for each treatment combination. An average of 75 seeds
was sown onto each plate for an average of 375 seeds per
treatment. Cultures were placed under a 12/12 h light/dark
photoperiod and cool-white fluorescent lights with a light level
of 40 μmol m−2 s−1. Cultures under fluctuating temperatures
Table 1. Population locations
and seed viability information
for Calopogon tuberosus seed
sources used in the present study
a
Based on a TTC test for embryo
viability using seed collected from
2007. Percentages represent the
mean response of two replications
of 100 seeds each (200 seeds total)
were placed in a Percival incubator (model 130VL, Percival
Scientific, Perry, IA), while cultures in constant temperatures
were placed in a Percival incubator model 136LL. Seed
germination and seedling development (Table 2) were
monitored bi-weekly for 8 wk according to the 6 developmental stages described by Kauth et al. (2006).
Cold stratification effects on germination. For this experiment, all populations except South Carolina 3 were used
because of lack of seed. After seed inoculation, cultures
were wrapped in aluminum foil and placed in complete
darkness at 10°C for 2, 4, 6, and 8 wk. A control of no chill
was also used. Ten degrees was used as the stratification
temperature because this temperature is commonly encountered throughout the range of C. tuberosus during colder
months (Fig. 1). Five replications were used for each seed
treatment combination. Approximately 60 seeds were sown
onto each plate for an average of 300 seeds per treatment.
Seed numbers were counted prior to cold stratification.
After each designated cold stratification period, vessels
were removed, unwrapped, and maintained in a 12/12 h
light/dark photoperiod under cool white fluorescent lights
Population
Habitat
Latitude
Michigan
South Carolina 1
South Carolina 2
South Carolina 3
North Central Florida
South Florida
Northern Fen
Fen
Granite Seepage
Cataract Bog
Roadside
Wet Prairie
26°
29°
35°
35°
35°
45°
10′
09′
05′
05′
05′
34′
06″
18″
13″
02″
03″
47″
Viable embryosa
Longitude
N
N
N
N
N
N
81°
82°
82°
82°
82°
87°
21′
37′
34′
35′
36′
39′
51″
12″
46″
51″
27″
38″
W
W
W
W
W
W
20.8%
25.0%
10.6%
30.3%
53.9%
70.5%
COMPARATIVE IN VITRO GERMINATION ECOLOGY OF CALOPOGON TUBEROSUS
Table 2. Six stages of Calopogon
tuberosus seed development (from
Kauth et al. 2006)
Stage
Description
0
1
2
3
4
5
6
Non-germinated seed
Imbibed seed, swollen and greening still covered or partially covered by testa
Enlarged seed without testa
Protocorm with pointed shoot apex and rhizoids
Protocorm with emerging leaf and developing rhizoids
Seedling with one elongated leaf and one developing root
Seedling with evident roots and two or more leaves
(40 μmol m−2 s−1) at 24.2±0.2°C. Culture vessels were
initially scored upon removal from the dark, cold stratification period. Germination and development were examined bi-weekly for 10 wk after culture vessels were
removed from cold storage. Cultures in the 2-wk chill were
under non-chilling conditions for 8 wks, 4-wk chill cultures
for 6 wk, 6-week chill cultures for 4 wk, and 8-week chill
cultures for 2 wk.
Data analysis. Germination percentages were calculated by
dividing the number of germinated seeds by the total
number of seeds in each replication. A developmental index
of germinated seeds was modified from (Otero et al. 2004):
DI ¼
151
ð N1 þ N2 2 þ N3 3 þ N 4 4 þ N 5 5 þ N6 6 Þ
ð N1 þ N2 þ N 3 þ N4 þ N5 þ N 6 Þ
where N1 is the number of seeds in stage 1, etc. Stage 0
ungerminated seeds were excluded since we were
concerned only with germinated seeds. All data were
arcsine transformed to normalize variation, but nontransformed means are presented. Germination and seedling
development data were analyzed using general linear model
procedures and least square means at α=0.05 in SAS v 9.1
(SAS Institute 2003).
Results
Effects of temperature on seed germination. Population
(F5 =410.4, p<0.0001), temperature (F4 =10.6, p<0.0001),
and their interaction (F20 =5.08, p<0.0001) all had a
significant effect on germination. In all populations, higher
germination percentages occurred in at least some fluctuating
temperatures compared to the constant 25°C (Fig. 2).
Significantly higher germination (>20%) in Michigan seeds
occurred at temperatures below 33/24°C with the exception
of 25°C (Fig. 2a). Higher fluctuating temperatures promoted
germination in all South Carolina populations. However,
germination in South Carolina 1 and 2 populations was still
less than 10% regardless of temperature (Fig. 2b, c). Higher
fluctuating temperatures (29/19°C and 33/24°C) promoted
germination in north central Florida seeds (Fig. 2e). Surprisingly, south Florida seed germination was highest at the
extremes of 22/11°C and 33/24°C (Fig. 2f). Among all
populations, germination was highest in south Florida seeds,
followed by north central Florida, South Carolina 3,
Michigan and South Carolina 1 and 2.
Effects of temperature on protocorm and seedling development. Population (F5 =79.6, p<0.0001), temperature (F4 =
9.83, p<0.0001), and their interaction (F20 =4.38, p<0.0001)
all had a significant effect on early development. Among
seed sources, development of germinated seeds was more
advanced in Michigan (Fig. 3a) and South Carolina 3
(Fig. 3d) seedlings with the majority near or above stage 5.
Above 22/11°C, development was more advanced among
temperature regimes in Michigan, South Carolina 3 and
north Central Florida (Fig. 3e). South Florida seedlings were
more developed at temperatures below 33/24°C (Fig. 3f).
Seedling development was more advanced above 22/11°C
and under all fluctuating temperatures (Fig. 3b). South
Carolina 2 seedlings were more developed at 33/24°C
(Fig. 3c).
Effects of cold stratification on germination. Because of
low germination in the temperature study, a coldstratification study was conducted to investigate possible
seed dormancy. Population (F4 =44.2, p<0.0001), chilling
period (F4 =264.4, p<0.0001), and their interaction (F16 =
22.4, p<0.0001) all had a significant effect on germination.
In general, longer chilling periods resulted in significantly
higher germination percentages after 10-wk culture, and the
lowest germination was observed in unchilled seeds
(Fig. 4). In fact, no germination was observed in the control
for South Carolina 2. In seeds from Michigan, chill periods
over 4 wk promoted the highest seed germination. In both
South Carolina populations, chilling seeds for 6 or 8 wk
promoted the highest germination. No differences among
chilling duration were observed in north central Florida
seeds, but germination of chilled seeds was significantly
higher than unchilled seeds. Higher germination in south
Florida seeds was observed in chilling periods of 4 wk or
152
KAUTH ET AL.
Figure 2. Temperature effects on
seed germination of Calopogon
tuberosus from different populations after 8 wk culture under a
12 h photoperiod. (a) Michigan
population (b) South Carolina
population 1 (c) South Carolina
population 2 (d) South Carolina
population 3 (e) North Central
Florida population (f) South
Florida population. Histobars
within each seed source with the
same letter are not significantly
different (α=0.05).
longer. In south Florida and South Carolina populations,
maximum germination was approximately 80% compared
to 70% and 65% germination in Michigan and north central
Florida seeds, respectively (Fig. 4).
Discussion
C. tuberosus seeds from diverse habitats responded differently to cold stratification and optimum temperature
regimes. The variability in responses to cold stratification
and different temperature regimes provide further insight
into ecotypic differentiation for temperature. Together with
our previous results (Kauth et al. 2008b; Kauth and Kane
2009), these experiments provide the groundwork for using
in vitro ecology studies to differentiate ecotypes.
Because clear differences in temperature responses in
Florida seeds were not as evident compared to Michigan
and South Carolina seeds, they may be more responsive to
other environmental cues such as photoperiod or chemical
signals from mycorrhizal fungi (Baskin and Baskin 2001).
Differences in cold stratification requirement among seed
sources could be influenced by temperatures experienced
by the mother plant during seed maturation (Ballard 1987)
or indicate varying degrees of seed dormancy.
Embryo viability and germination. Results from the TTC
test should be interpreted with caution, because embryo
viability is often inaccurate due to testa impermeability
(Lauzer et al. 1994; Vujanovic et al. 2000). TTC tests also
do not account for certain enzymes that become active
during germination (Lauzer et al. 1994). However, TTC
testing provided a fairly accurate estimate of embryo
viability in the Florida populations, but not in the Michigan
and South Carolina seeds. If the germination percentages
from the diurnal temperature experiment are accounted for,
embryo viability was accurate. Because cold stratification
increased germination, embryo viability was higher in
Michigan and South Carolina seeds compared to the TTC
results. Given that cold stratification increased germination,
and any seed that germinates is viable, the cold stratification data more accurately measured embryo viability. Large
discrepancies between germination and embryo viability in
seeds from northern populations may be due to differences
in testa permeability. The 3-h pretreatment in CaOCl2 may
COMPARATIVE IN VITRO GERMINATION ECOLOGY OF CALOPOGON TUBEROSUS
153
Figure 3. Temperature effects
on seedling development of Calopogon tuberosus from different
populations after 8 wk culture
under a 12 h photoperiod. Development index of describes the
average stage of development of
germinated seeds. (a) Michigan
population (b) South Carolina
population 1 (b) South Carolina
population 2 (c) South
Carolina population 3 (e) North
Central Florida population (f)
South Florida population.
Histobars within each seed
source with the same letter are
not significantly different
(α=0.05).
not have been an optimal duration to degrade the testa in
seeds from northern populations to achieve maximum
embryo staining. A thicker testa could be an adaptation to
protect the embryo from colder temperatures.
Temperature effects. In general, the highest germination
percentages occurred under fluctuating temperatures rather than
a constant temperature. As constant temperatures are not
normally encountered by seeds in situ (Baskin et al. 2006),
the decrease in germination observed at 25°C is not surprising.
Previous research with non-orchids indicated that fluctuating
diurnal temperatures promoted germination rather than constant temperatures (Thompson et al. 1977; Thompson and
Grime 1983; Probert et al. 1986). Seed germination of C.
tuberosus still occurred at 25°C, which may reflect the
removal of fluctuating temperature requirements in the
presence of light (Thompson and Grime 1983). However,
fluctuating temperatures have been shown to enhance germination in non-orchid seeds exposed to light (Toole et al. 1955).
Increased germination of orchid seeds at constant temperatures may be caused by germination and mycorrhizal fungi
infection at deeper soil depths, especially in species that
germinate poorly in light (Rasmussen and Rasmussen 1991).
C. tuberosus seeds, conversely, germinate in both light and
dark (Stoutamire 1974; Kauth et al. 2006), suggesting that C.
tuberosus seeds can germinate near the soil surface to utilize
fluctuating temperatures, light and higher nitrate concentrations (Roberts and Benjamin 1979). As temperature
fluctuations occur more often near the soil surface, increased
germination under fluctuating temperatures is a mechanism
to prevent germination of deeply buried seeds (Thompson
and Grime 1983). However, higher germination at 25°C of
south Florida seeds may be a consequence of burial. Some
seeds may not germinate at the soil surface, but rather
germinate slowly underneath the soil and emerge under
cooler temperatures (Probert et al. 1985).
Germination of South Carolina seeds at 33/24°C was
rather surprising. The habitats of all South Carolina
populations are wetland areas with seasonal water flow
(personal observation). Certain wetland species have been
shown to require high fluctuating temperatures and light to
maximize germination (Thompson and Grime 1983). As the
water table falls in wetlands, gaps are created; the
surrounding soil heats and seeds are exposed to light
154
KAUTH ET AL.
Figure 4. Effects of chilling seeds at 10°C in darkness on germination of
Calopogon tuberosus from distant populations after 10-wk culture. (a)
Michigan population (b) South Carolina population 1 (c) South Carolina
population 2. (d) North Central Florida population (e) South Florida
population. Cultures were incubated under a 12 h photoperiod and 24°C
once removed from the chilling treatment. Cultures in the 2-wk chill
were under non-chilling conditions for 8 wk, 4-wk chill cultures for
6 wk, 6-wk chill cultures for 4 wk and 8-wk chill cultures for 2 wk.
Control cultures were not chilled and incubated for 10 wk. Histobars
within with the same letter are not significantly different (α=0.05).
(Thompson and Grime 1983). Because the soil is shallow
(often less than 2 cm deep, personal observation) in the South
Carolina populations, the soil can heat rapidly when exposed
to light, and seeds are subsequently exposed to higher
temperature fluctuations (van Assche and Vanlerberghe
1989). The response of south Florida seeds to both the
lowest and highest fluctuating temperature regime was
surprising as well, indicating that these seeds may require
warm followed by cold stratification to break dormancy
(Rasmussen 1992; Hidayati et al. 2000).
Cold stratification effects. Chilling increased germination
significantly compared to the control among all C. tuberosus
seed populations. This is not surprising because chilling has
been reported to increase germination in seeds of numerous
orchid species (De Pauw and Remphrey 1993; Chu and
Mudge 1994; Tomita and Tomita 1997; Miyoshi and Mii
1998; Shimura and Koda 2004, 2005; Øien et al. 2008). The
results from this study indicate that physiological dormancy
is present in seeds from several C. tuberosus populations.
The embryos may not be completely dormant, but the testa is
likely inducing dormancy (Lauzer et al. 2007).
Although the exact mechanism of chilling in orchid
seeds is not well understood, chilling likely breaks
physiological dormancy (Baskin and Baskin 2001; Øien et
al. 2008). A chilling requirement would prevent germination immediately after capsule dehiscence when environmental conditions are not adequate and mycorrhizal fungi
needed for germination are not active (Rasmussen 1995;
Baskin and Baskin 2001). Although a constant temperature
was used after the chill period, previous research has
indicated that chilling may remove a requirement for
fluctuating temperatures (van Assche et al. 2003). Concerns
that surface sterilizing seeds may weaken the seed coat and
lead to higher germination are not compelling because
germination was still lower in the non-chilled seeds.
The degree of dormancy is also likely different among
populations because Michigan seeds did not germinate to
the maximum percentages observed in other populations.
Seeds from northern populations may require longer cold
stratification that delay germination until late winter or
spring to break deeper dormancy (Meyer et al. 1995; Allen
and Meyer 1998) or further degrade a thicker testa that
protect seeds from colder temperatures. Seeds from northern climates may also require a colder chilling temperature
to further break dormancy. Investigating the role of
temperature on chilling would provide information on
whether southern seeds could tolerate colder temperatures.
The positive response to cold stratification of Florida
seeds was surprising. Although Florida seeds encounter
below freezing temperatures in nature, the cold treatment is
not necessary for germination, but seeds still respond to
cold stratification (Nakamura 1964). However, an ecological adaptation to a cold stratification requirement in Florida
seeds cannot be overlooked. Southern populations may
require a chilling period because warm winter temperatures
may lead to early seedling emergence and subsequent death
from sudden cold temperatures (Fowler and Dwight 1964;
Schütz and Milberg 1997). The required chilling period
may ensure that seeds germinate only upon experiencing
temperatures that would not harm emerging seedlings.
Nondormant seeds from south Florida germinate slowly
with little development indicating that autumn emergence is
unlikely (Meyer et al. 1995). South Florida seeds likely are
not exposed to a long chilling period, which can cause a
breakdown of chilling cue resistance so seeds can germinate
(Meyer 1992).
The results of this study provide insight into the nature
of orchid seed ecology and physiology, and specifically
germination requirements within a widespread species. To
advance our understanding about orchid seed germination
ecology, using the framework from non-orchid seed studies
provides meaningful insight. When interpreting in vitro
data, we must be careful since in vitro studies like other ex
situ conditions can oversimplify selection pressures since in
vitro and in situ conditions differ greatly. Selection
pressures do not act alone, but photoperiod, temperature,
soil nutrients and type, pollination events and water
availability interact and form a complex network directing
ecotypic differentiation. Although in situ germination has
not been studied in C. tuberosus, field germination studies
may reveal important ecological data such as timing of
germination. However, this study provides framework to
COMPARATIVE IN VITRO GERMINATION ECOLOGY OF CALOPOGON TUBEROSUS
further study the germination ecology of C. tuberosus and
other orchid species.
Acknowledgments We thank the following for collecting seed:
Larry Richardson (Florida Panther National Wildlife Refuge, Naples,
FL), Jim Fowler (South Carolina populations), and Kip Knudson
(Michigan population). We also thank Mary Bunch (South Carolina
Heritage Preserve Program) for issuing collection permits. We thank
Dr. Hector Perez (University of Florida, Gainesville, FL) for use of
growth chambers. We also thank Timothy Johnson for providing
insightful comments regarding the manuscript. Brand names are
provided as references; the authors do not solely recommend or
endorse these products. US Fish and Wildlife and the Florida Panther
National Wildlife Refuge provided partial funding.
References
Allen P. S.; Meyer S. E. Ecological aspects of seed dormancy loss.
Seed Sci. Res. 8: 183–191; 1998.
Ballard W. W. Sterile propagation of Cypripedium reginae from seeds.
Am. Orchid Soc. Bull. 56: 935–946; 1987.
Baskin C. C.; Baskin J. M. Seeds: ecology, biogeography, and evolution
of dormancy and germination. Academic, San Diego; 2001.
Baskin C. C.; Baskin J. M. Determining dormancy-breaking and
germination requirements from the fewest seeds. In: Guerrant E.
O.; Havens K.; Maunder M. (eds) Ex situ plant conservation:
supporting species survival in the wild. Island Press, Washington,
pp 162–179; 2004.
Baskin C. C.; Thompson K.; Baskin J. M. Mistakes in germination
ecology and how to avoid them. Seed Sci. Res. 16: 165–168; 2006.
Bewley J. D.; Black M. Seeds: physiology of development and
germination. Plenum, New York; 1994.
Brundrett M. C.; Scade A.; Batty A. L.; Dixon K. W.; Sivasithamparam
K. Development of in situ and ex situ seed baiting techniques to
detect mycorrhizal fungi from terrestrial orchid habitats. Mycol.
Res. 107: 1210–1220; 2003.
Chu C. C.; Mudge K. W. Effects of prechilling and liquid suspension culture
on seed germination of the yellow lady's slipper orchid (Cypripedium
calceolus var. pubescens). Lindleyana 9: 153–159; 1994.
De Pauw M. A.; Remphrey W. R. In vitro germination of three
Cypripedium species in relation to time of seed collection, media,
and cold treatment. Can. J. Bot. 71: 879–885; 1993.
Diez J. M. Hierarchical patterns of symbiotic orchid germination
linked to adult proximity and environmental gradients. J. Ecol.
95: 159–170; 2007.
Donohue K. Seeds and seasons: interpreting germination timing in the
field. Seed Sci. Res. 15: 175–187; 2005.
Fowler D. P.; Dwight T. W. Provenance differences in the stratification
requirements of white pine. Can. J. Bot. 42: 669–675; 1964.
Goldman D. H.; Jansen R. K.; van den Berg C.; Leitch I. J.; Fay M. F.;
Chase M. W. Molecular and cytological examination of Calopogon
(Orchidaceae, Epidendroideae): circumscription, phylogeny, polyploidy, and possible hybrid speciation. Am. J. Bot. 91: 707–723;
2004a.
Goldman D. H.; van den Berg C.; Griffith M. P. Morphometric
circumscription of species and infraspecific taxa in Calopogon R.
Br. (Orchidaceae). Plant Syst. Evol. 247: 37–60; 2004b.
Harvais G. Growth requirements and development of Cypripedium
reginae in axenic culture. Can. J. Bot. 51: 327–332; 1973.
Hidayati S. N.; Baskin C. C.; Baskin J. M. Dormancy-breaking and
germination requirements of seeds of four Lonicera species
155
(Caprifoliaceae) with underdeveloped spatule embryos. Seed Sci.
Res. 10: 459–469; 2000.
Hufford K. M.; Mazer S. J. Plant ecotypes: genetic differentiation in the
age of ecological restoration. Trends Ecol. Evol. 18: 147–155; 2003.
Kauth P. J.; Dutra D.; Johnson T. R.; Stewart S. L.; Kane M. E.;
Vendrame W. A. Techniques and applications of in vitro orchid
seed germination. In: Teixeira da Silva J. A. (ed) Floriculture,
ornamental and plant biotechnology: advances and topical issues.
Global Science Books, Isleworth, pp 375–391; 2008a.
Kauth P. J.; Kane M. E. In vitro ecology of Calopogon tuberosus var.
tuberosus (Orchidaceae) seedlings from distant populations:
implications assessing for ecotypic differentiation. J. Torrey
Bot. Soc. 136: 433–444; 2009.
Kauth P. J.; Kane M. E.; Vendrame W. A.; Reinhardt-Adams C.
Asymbiotic germination response to photoperiod and nutritional
media in six populations of Calopogon tuberosus var. tuberosus
(Orchidaceae): evidence for ecotypic differentiation. Ann. Bot.
102: 783–793; 2008b.
Kauth P. J.; Vendrame W. A.; Kane M. E. In vitro seed culture and
seedlings development of Calopogon tuberosus. Plant Cell
Tissue Organ Cult. 85: 91–102; 2006.
Lauzer D.; Renaut S.; St-Arnaud M.; Barabe D. In vitro asymbiotic
germination, protocorm development, and plantlet acclimatization of Aplectrum hyemale (Muhl. ex Willd.) Torr. (Orchidaceae).
J. Torrey Bot. Soc. 134: 344–348; 2007.
Lauzer D.; St-Arnaud M.; Barabe D. Tetrazolium staining and in vitro
germination of mature seeds of Cypripedium acaule (Orchidaceae).
Lindleyana 9: 197–204; 1994.
Luer C. A. The native orchids of Florida. The New York Botanical
Garden, New York; 1972.
McKay J. K.; Christian C. E.; Harrison S.; Rice K. J. “How local is
local?”—a review of practical and conceptual issues in the
genetics of restoration. Restor. Ecol. 13: 432–440; 2005.
Meyer S. E. Habitat correlated variation in firecracker penstemon
(Penstemon eatonii Gray: Scrophulariaceae) seed germination
response. Bull. Torrey Bot. Club 119: 268–279; 1992.
Meyer S. E.; Kitchen S. G.; Carlson S. L. Seed germination timing
patterns in intermountain Penstemon (Scrophulariaceae). Am. J.
Bot. 82: 377–389; 1995.
Miyoshi K.; Mii M. Stimulatory effects of sodium and calcium
hypochlorite, pre-chilling and cytokinins on the germination of
Cypripedium macranthos seed in vitro. Physiol. Plant. 102: 481–
486; 1998.
Mweetwa A. M.; Welbaum G. E.; Tay D. Effects of development,
temperature, and calcium hypochlorite treatment on in vitro
germinability of Phalaenopsis seeds. Sci. Hortic. 117: 257–262;
2008.
Nakamura S. I. Einige experimente zur samenkeimung einer chlorophyllfreien Erodorchidee Galeola septentrionalis Reichb. f. Mem. Coll.
Agric., Kyoto Univ. 86: 1–48; 1964.
Øien D. I.; O'Neill J. P.; Whigham D. F.; McCormick M. K.
Germination ecology of the boreal-alpine terrestrial orchid
Dactylorhiza lapponica (Orchidaceae). Ann Bot Fenn 45: 161–
172; 2008.
Otero J. T.; Ackerman J. D.; Bayman P. Differences in mycorrhizal
preferences between two tropical orchids. Mol. Ecol. 13: 2393–
2404; 2004.
Probert R. J.; Smith R. D.; Birch P. Germination responses to light and
alternating temperatures in European populations of Dactylis
glomerata L. I. variability in relation to origin. New Phytol. 99:
305–316; 1985.
Probert R. J.; Smith R. D.; Birch P. Germination responses to light
and alternating temperatures in European populations of
Dactylis glomerata L.5. The principle components of the
alternating temperature requirement. New Phytol. 102: 133–
142; 1986.
156
KAUTH ET AL.
Rasmussen H. N. Seed dormancy patterns in Epipactis palustris
(Orchidaceae): requirements for germination and establishment
of mycorrhiza. Physiol. Plant. 86: 161–167; 1992.
Rasmussen H. N. Terrestrial orchids from seed to mycotropic plant.
Cambridge University Press, Cambridge; 1995.
Rasmussen H. N.; Andersen T. F.; Johansen B. Temperature
sensitivity of in vitro germination and seedling development of
Dactylorhiza majalis (Orchidaceae) with and without a mycorrhizal fungus. Plant Cell Environ. 13: 171–177; 1990.
Rasmussen H. N.; Rasmussen F. N. Climactic and seasonal
regulation of seed plant establishment in Dactylorhiza majalis
inferred from symbiotic experiments in vitro. Lindleyana 5:
221–227; 1991.
Roberts E. H.; Benjamin S. K. The interaction of light, nitrate and
alternating temperature on the germination of Chenopodium
album, Capsella bursa-pastoris and Poa annua before and after
chilling. Seed Sci. Technol. 7: 379–392; 1979.
SAS Institute. SAS for Windows, version 9.1. Cary, NC, USA; 2003.
Schütz W.; Milberg P. Seed dormancy in Carex canescens: regional
differences and ecological consequences. Oikos 78: 420–428;
1997.
Seneca E. D. Germination and seedling response of Atlantic and Gulf
coast populations of Uniola paniculata. Am. J. Bot. 59: 290–296;
1972.
Seneca E. D. Germination and seedling response of Atlantic and Gulf
coasts populations of Spartina alterniflora. Am. J. Bot. 61: 947–
956; 1974.
Shimura H.; Koda Y. Micropropagation of Cypripedium macranthos
var. rebunense through protocorm-like bodies derived from
mature seeds. Plant Cell Tissue Organ Cult. 78: 273–276; 2004.
Shimura H.; Koda Y. Enhanced symbiotic seed germination of
Cypripedium macranthos var. rebunense following inoculation
after cold treatment. Physiol. Plant. 123: 281–287; 2005.
Singh K. P. Effect of temperature and light on seed germination of two
ecotypes of Portulaca oleracea L. New Phytol. 72: 289–295;
1973.
Stoutamire W. P. Terrestrial orchid seedlings. In: Withner C. L. (ed)
The orchids: scientific studies. Wiley, New York, pp 101–128;
1974.
Thompson K.; Grime J. P. A comparative study of germination responses to
diurnally fluctuating temperatures. J. Appl. Ecol. 20: 141–156; 1983.
Thompson K.; Grime J. P.; Mason G. Seed germination in response to
diurnal fluctuations of temperature. Nature 267: 147–149; 1977.
Tomita M.; Tomita M. Effects of culture media and cold treatment on
germination in asymbiotic culture of Cypripedium macranthos
and Cypripedium japonica. Lindleyana 12: 208–213; 1997.
Toole E. H.; Toole V. K.; Borthwick H. A.; Hendricks S. B.
Interaction of temperature and light in germination of seeds.
Plant Physiol. 30: 473–478; 1955.
Trapnell D. W.; Hamrick J. L.; Giannasi D. E. Genetic variation and
species boundaries in Calopogon (Orchidaceae). Syst. Bot. 29:
308–315; 2004.
van Assche J. A.; Debucquoy K. L. A.; Rommens W. A. F. Seasonal
cycles in the germination capacity of buried seeds of some
Leguminosae (Fabaceae). New Phytol. 158: 315–323; 2003.
van Assche J. A.; Vanlerberghe K. A. The role of temperature on the
dormancy cycle of seeds of Rumex obtusifolius L. Funct. Ecol. 3:
107–115; 1989.
van Waes J. M.; Debergh P. C. In vitro germination of some Western
European orchids. Physiol. Plant. 67: 253–261; 1986.
Vujanovic V.; St-Arnaud M.; Barabe D.; Thibeault G. Viability testing
of orchid seed and the promotion of colouration and germination.
Ann. Bot. 86: 79–86; 2000.
Zettler L. W.; Piskin K. A.; Stewart S. L.; Hartsock J. J.; Bowles M.
L.; Bell T. J. Protocorm mycobionts of the federally threatened
eastern prairie fringed orchid, Platanthera leucophaea (Nutt.)
Lindley, and a technique to prompt leaf elongation in seedlings.
Stud. Mycol. 53: 163–171; 2005.
Zettler L. W.; Stewart S. L.; Bowles M. L.; Jacobs K. A. Mycorrhizal
fungi and cold-assisted symbiotic germination of the federally
threatened eastern prairie fringed orchid, Platanthera leucophaea
(Nuttall) Lindley. Am. Midl. Nat. 145: 168–175; 2001.