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EFFECTS OF CARBOHYDRATE SOURCE
ON THE IN VITRO ASYMBIOTIC SEED
GERMINATION OF THE TERRESTRIAL
ORCHID HABENARIA MACROCERATITIS
Scott L. Stewart
a b
& Michael E. Kane
a
a
Plant Restoration, Conservation, and Propagation Biotechnology
Program, Environmental Horticulture Department, University of
Florida, Gainesville, Florida, USA
b
Horticulture and Agriculture Programs, Kankakee Community
College, Kankakee, Illinois, USA
Available online: 13 May 2010
To cite this article: Scott L. Stewart & Michael E. Kane (2010): EFFECTS OF CARBOHYDRATE
SOURCE ON THE IN VITRO ASYMBIOTIC SEED GERMINATION OF THE TERRESTRIAL ORCHID HABENARIA
MACROCERATITIS , Journal of Plant Nutrition, 33:8, 1155-1165
To link to this article: http://dx.doi.org/10.1080/01904161003763757
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Journal of Plant Nutrition, 33:1155–1165, 2010
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Copyright ISSN: 0190-4167 print / 1532-4087 online
DOI: 10.1080/01904161003763757
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EFFECTS OF CARBOHYDRATE SOURCE ON THE IN VITRO
ASYMBIOTIC SEED GERMINATION OF THE TERRESTRIAL ORCHID
HABENARIA MACROCERATITIS
Scott L. Stewart1,2 and Michael E. Kane1
1
Plant Restoration, Conservation, and Propagation Biotechnology Program, Environmental
Horticulture Department, University of Florida, Gainesville, Florida, USA
2
Horticulture and Agriculture Programs, Kankakee Community College, Kankakee,
Illinois, USA
2
The effects of the carbohydrates fructose, sucrose, and dextrose on the seed germination and
protocorm development of the terrestrial orchid Habenaria macroceratitis were assessed in the
presence or absence of banana powder. Malmgren Modified Terrestrial Orchid Medium was used as
the basal medium. Each carbohydrate was screened at 50 mM with and without banana powder
(15 g L−1). No significant differences in germination or protocorm development among all treatments were observed after seven weeks. There were no significant differences in advanced development among carbohydrate treatments without banana powder after 21 weeks. However, the presence of banana powder in the medium suppressed advanced protocorm development in the fructose,
dextrose, and control treatments. These data support the wide suitability of simple exogenous carbohydrates in supporting the germination and protocorm development of H. macroceratitis, and
suggest that a wide range of carbohydrates are suitable for the culture of often difficult-to-germinate
terrestrial orchids.
Keywords: asymbiotic, carbohydrate, dextrose, fructose, Habenaria macroceratitis, Orchidaceae, seed germination, sucrose, terrestrial orchid
INTRODUCTION
Asymbiotic orchid seed germination is one of the most prevalent methods of orchid plant production in commercial settings and has been highly
favoured by scientific researchers as a tool to study orchid seed germination
physiology and early life stages (Kauth et al., 2008). Knudson (1916, 1922,
1924), Clement (1924, 1926), and Ballion and Ballion (1924) were among
Received 10 August 2008; accepted 17 June 2009.
Address correspondence to Scott L. Stewart, Horticulture and Agriculture Programs, Kankakee
Community College, Kankakee, IL 60901, USA. E-mail: sstewart@kcc.edu
1155
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S. L. Stewart and M. E. Kane
the first researchers to establish the method of in vitro asymbiotic orchid
seed germination and recognize the role macro- and micro-nutrients and,
especially, simple carbohydrates played in the germination process of orchid
seed. In particular, Knudson (1922) was the first to demonstrate that orchid
seed could be germinated in vitro on a defined nutrient medium supplemented with a simple carbohydrate source (sucrose). Until this discovery it
was thought that orchid seed could only be germinated in co-culture with
an appropriate mycorrhizal fungus (= symbiotic seed germination; Wynd,
1933). Today, asymbiotic orchid seed germination has become the standard
for commercial orchid production and research on orchid seed germination
physiology.
Many researchers have used asymbiotic orchid seed germination as
a means to study various aspects of orchid seed germination physiology
and protocorm/seedling nutrition. Some topics examined using asymbiotic
methods include: in vitro mineral nutrition (Stenberg and Kane, 1998; Spoerl
and Curtis, 1948; Kauth et al., 2006), effects of exogenous plant growth regulators (Stewart and Kane, 2006; Miyoshi and Mii, 1998; de Pauw et al., 1995),
effects of photoperiod (Johnson and Kane, 2007; Stewart and Kane, 2006;
Dutra et al., 2008), and as a comparative germination method to symbiotic
orchid seed germination (Johnson et al., 2007). The effects of carbohydrate
source have long been studied in relation to asymbiotic orchid seed germination; however, despite this long history little practical production, growth,
and development work has been published in this area.
The role of carbohydrates in in vitro orchid seed germination and protocorm/seedling growth has received some attention, although little in recent
years. Early in the study of asymbiotic orchid seed germination, researchers
realized that some carbohydrate sources were better suited to support seed
germination than others, and that this response was genus- or species-specific
(La Garde, 1929; Smith, 1932; Wynd, 1933). Ernst et al. (1971) and Ernst and
Arditti (1990) demonstrated that a wide range of carbohydrates, such as glucose, fructose, and oligosaccharides containing these sugars, were suitable to
support the growth and development of Phalaenopsis seedlings under in vitro
conditions. Similarly, asymbiotic methods have been used to demonstrate
the rapid uptake of simple sugars (fructose) by both differentiated and undifferentiated Dendrobium tissues (Hew and Mah, 1989). Harrison and Arditti
(1978) used asymbiotic germination methods to investigate the role of carbohydrate source in the germination of the epiphytic orchid Cattleya aurantiaca.
Using the fungal carbohydrates trehalose and mannitol, as well as glucose,
Smith (1973) demonstrated that seed of the terrestrial orchids Dactylorhiza
purpurella and Bletilla striata utilized trehalose and glucose as suitable primary
carbohydrate sources during asymbiotic germination, and utilized mannitol
to a lesser extent. Finally, examining in vitro seed germination of both Phalaenopsis Habsburg and Phalaenopsis Ruth Burton × (Phalaenopsis Abendrot
× Phalaenopsis Abendrot), Ernst and Arditti (1990) reported that a number
Carbohydrate Effects on Orchid Seed Germination
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of carbohydrate sources, glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose, were all individually capable
of supporting seed germination.
Little attention has been paid by modern researchers to the effects
of carbohydrate source on asymbiotic orchid seed germination and protocorm/seedling development, and little information exists on the role of
different carbohydrates in the asymbiotic germination of terrestrial orchids.
In the present study, we provide new information on the effects of simple
carbohydrates, fructose, sucrose, and dextrose, and the medium supplement
banana powder on the asymbiotic seed germination and protocorm development of the terrestrial orchid Habenaria macroceratitis.
MATERIALS AND METHODS
Seed Source and Sterilization
Habenaria macroceratitis seeds were obtained from mature capsules collected prior to dehiscence on from a population in Hernando County,
Florida. Immediately after collection, capsules were dried over silica gel desiccant for two weeks at 25 ± 5◦ C, followed by storage in darkness at −20◦ C for
52 days. Seeds were surface disinfected for 1 min in a solution containing 5
mL ethanol (100%), 5 mL 6.00% sodium hypochloride (NaOCl), and 90 mL
sterile deionized (DI) water. Following surface disinfection, seeds were
rinsed three times for 1 min each in sterile DI water. Solutions were removed from the surface disinfection vial using a sterile Pasture pipette that
was replaced after each use. Sterile DI water was used to suspend the disinfected seed, and a sterile bacterial inoculating loop was used to sow the seed.
An average of 210 seeds per petri plate were sown.
Effects of Carbohydrate Source on Asymbiotic Seed Germination
Effects of three carbohydrate sources [fructose, sucrose, and dextrose
(Sigma-Aldrich, St. Louis, MO, USA)] at 50 mM on the asymbiotic seed germination and protocorm development of H. macroceratitis were examined.
Malmgren Modified Terrestrial Orchid Medium (MM; PhytoTechnology
Laboratories, Shawnee Mission, KS, USA) was used in treatment combinations with carbohydrate source and the presence or absence of banana powder (BP; PhytoTechnology Laboratories, Shawnee Mission, KS, USA). Both
the carbohydrate concentration (S.L. Stewart, unpublished data) and asymbiotic germination medium (Stewart and Kane, 2006) were chosen based on
preliminary studies (data not shown). The medium was prepared using standard methods (Stewart and Kane, 2006) and supplemented with a 50 mM
carbohydrate source (9.008 g L−1 fructose, 17.115 g L−1 sucrose, 9.008 g L−1
dextrose). To those treatments containing BP, 15 g L−1 BP was added prior to
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S. L. Stewart and M. E. Kane
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sterilization. Medium pH was adjusted to 5.8 using 1.0 N potassium hydroxide (KOH) prior to autoclaving for 20 min at 121◦ C and 117.7 kPa. Sterile
medium was dispensed (ca. 25 mL) into 9 cm diameter petri plates. Ten
replicate plates were inoculated with seed. Plates were sealed with one layer
of Nescofilm. Seed germination and protocorm development were scored after 7 and 21 weeks dark incubation at 25◦ C ± 2◦ C. Germination percentages
and statistical analyses were completed using general linear model procedures and least square means at α = 0.05 (SAS, Version 8.02, SAS Institute,
Cary, NC, USA). Seed germination percentages were arcsine transformed
prior to analysis.
RESULTS
After seven weeks dark incubation, all carbohydrate sources tested supported at least minimal asymbiotic germination (≥Stage 1) of H. macroceratitis seeds. Stage 3 protocorm development was supported at this time by
both fructose without banana powder and basal medium only control treatments (0.2% and 0.4%, respectively; Figure 1). However, the majority of seed
was observed in Stage 2 development, with the basal medium only control
(83.9%), basal medium with banana powder control (81.0%), dextrose without banana powder (80.9%), and fructose without banana powder (79.1%)
supporting the highest developmental percentages in this developmental
stage (Figure 1). Only the fructose with banana powder treatment supported
a significantly lower protocorm development percentage (57.6%) than did
all other treatments. No significant difference in germination and developmental percentages were found for Stage 1 germination in all treatments
after seven weeks.
All carbohydrate sources supported seed germination and protocorm
development through Stage 5 development (Table 1) after 21 weeks in vitro
culture under dark incubation conditions. Both the basal medium only control and fructose without banana powder supported 100% Stage 5 development after 21 weeks (Figure 2). Only dextrose with banana powder supported a significantly lower protocorm developmental percentage in Stage
TABLE 1 Seed germination and protocorm development stages in
Habenaria macroceratitis, from Stewart and Kane (2006).
Stage
0
1
2
3
4
5
Description
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
1159
Carbohydrate Effects on Orchid Seed Germination
100
A
+ BP
- BP
Germination (%)
80
60
40
a
a
a
a
a
a
a
a
0
100
B
Germination (%)
80
60
40
20
a
a
a
a
a
a
a
a
0
100
C
b
b
ab
80
Germination (%)
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20
ab
b
b
ab
a
60
40
20
0
Fructose
Sucrose
Dextrose
Control
Carbohydrate Treatment
FIGURE 1 Effects of carbohydrate source (fructose, sucrose, and dextrose) and presence or absence
of banana powder (+/− BP) on percent germination and protocorm development of Habenaria macroceratitis. (A) Un-germinated seed (Stage 0), (B) initial seed germination (Stage 1), and (C) protocorm
development through Stage 2 after 7 week in vitro asymbiotic culture on Malmgren Modified Terrestrial
Orchid Medium. Histobars with the same letter within each stage are not significantly different (α =
0.05). Error bar = SE.
1160
S. L. Stewart and M. E. Kane
A
100
Germination (%)
80
60
40
20
a
B
Germination (%)
80
60
40
c
20
bc
bc
b
ab
a
0
100
c
80
c
bc
abc
bc
abc
Germination (%)
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a
0
100
C
ab
a
60
40
20
0
Fructose
Sucrose
Dextrose
Control
Carbohydrate Treatment
FIGURE 2 Effects of carbohydrate source (fructose, sucrose, and dextrose) and presence or absence of
banana powder (+/− BP) on percent germination and protocorm development of Habenaria macroceratitis. Protocorm development through (A) Stage 3, (B) Stage 4, and (C) Stage 5 after 21 weeks in vitro
asymbiotic culture on Malmgren Modified Terrestrial Orchid Medium. Histobars with the same letter
within each stage are not significantly different (α = 0.05). Error bar = SE.
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Carbohydrate Effects on Orchid Seed Germination
1161
5 (59.8%) than did all other treatments. This same treatment supported a
significantly higher percentage of Stage 4 protocorms (28.1%) than did all
other treatments tested. Furthermore, only the dextrose with banana powder
and fructose with banana powder treatments supported Stage 3 development
after 21 weeks (1.9% and 5.0%, respectively; Figure 2).
In the first seven weeks of asymbiotic seed germination, no significant
effect of medium supplementation with banana powder was demonstrated
in any treatment through Stage 2 development (Figure 1). However, after 21
weeks incubation the presence of banana powder in the medium suppressed
development of H. macroceratitis protocorms through Stage 5 in the fructose
and dextrose treatments (Figure 2). No significant difference in protocorm
development at Stage 5 was observed in the sucrose or control treatments.
Similar protocorm morphologies were observed in all carbohydrate and
carbohydrate plus BP treatments after 21 weeks in vitro germination and
development (Figures 3A–F). Protocorms in these treatments produced copious amounts of rhizoids in advanced (>Stage 4) developmental stages.
However, protocorms in both the basal medium only and basal medium
only plus BP control treatments produced either no or very diminished
rhizoids (Figures 3G–H).
DISCUSSION
In the current study, the simple carbohydrates fructose, sucrose, and
dextrose all supported germination and advanced (>Stage 3) protocorm
development of H. macroceratitis, demonstrating the wide suitability of simple
carbohydrates during in vitro asymbiotic germination and protocorm development. Similarly, Ernst and Arditti (1990) reported that seeds of the two
previously mentioned Phalaenopsis hybrids germinated rapidly on Knudson
C asymbiotic medium supplemented with glucose, maltose, or maltotriose,
which are also all simple carbohydrates. When larger maltooligosaccharides
where used, seed germination percentages decreased, likely due to the inability of the germinating orchid embryo to synthesize the enzymes necessary
to hydrolyze these more complex carbohydrates (Ernst and Arditti, 1990). A
more recent report indicated that carbohydrate hydrolysis by extracellular
hydrolytic enzymes is possible, as demonstrated with protocorm-like bodies
of Dendrobium (Hew and Mah, 1989). While this extracellular hydrolysis of
complex carbohydrates was not reported in the present study, a similar trend
in decreased seed germination percentage would be expected if larger, complex carbohydrates would be surveyed for their ability to support the asymbiotic germination of H. macroceratitis. The current data support the notion
that most orchids can readily utilize simple exogenous carbohydrates, as opposed to more complex carbohydrate sources, as a basic carbon source to
S. L. Stewart and M. E. Kane
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1162
FIGURE 3 Media effects on protocorm morphology of Habenaria macroceratitis seeds cultured on Malmgren Modified Terrestrial Orchid Medium supplemented with various carbohydrate sources and with
and without banana powder (BP) after 21 weeks. (A) Fructose + BP; (B) Fructose only; (C) Sucrose +
BP; (D) Sucrose only; (E) Dextrose + BP; (F) Dextrose only; (G) Control + BP; (H) Control only. Scale
bars = 1 mm.
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Carbohydrate Effects on Orchid Seed Germination
1163
initiate seed germination and support early protocorm development under
in vitro conditions.
While all carbohydrate treatments in the current study supported asymbiotic seed germination and protocorm development through Stage 5, both
controls (basal medium only and basal medium only with BP) also supported protocorm development through Stage 5. A similar trend has not
been previously reported in earlier studies concerning the effects of carbohydrate source on asymbiotic seed germination and in vitro protocorm
development. A minimal, but not quantified, amount of carbohydrate is
present in BP, and the advanced development observed in the basal medium
with BP treatment could be explained as being supported by this undefined
carbohydrate source. However, Stage 5 development was observed in the
basal medium without BP control treatment and this advanced stage of
H. macroceratitis protocorm development could not be supported without
additional exogenous carbohydrates. The basal medium (MM) contains
0.05 mg l−1 biotin and 100 mg l−1 myo-inositol, and these supplements could
serve as minimal sources of carbohydrate nutrition, in addition to BP. Arditti
(1979) reported that biotin enhanced the growth of Cattleya, Odontoglossum,
Phaphiopedilum, and Cymbidium orchids; and that inositol stimulated the germination of Cattleya. Both of these supplements in MM could be supporting
the germination through Stage 5 of H. macroceratitis in the basal medium
without BP control. Additionally, Rasmussen (1995) reported that some orchid seeds contain glucoproteins that may release glucose when hydrolyzed.
This too could also explain the advanced germination (=Stage 5) of
H. macroceratitis observed in the control treatments. Despite the stimulatory
effects of BP in the germination medium when used alone, BP in combination with a simple carbohydrate demonstrated either no or a negative effect
on asymbiotic seed germination percentages and protocorm development of
H. macroceratitis.
BP, in combination with an exogenous carbohydrate source, did effect
protocorm morphology, especially at more advanced stages (>Stage 4) of development. Media treatments containing only an exogenous carbohydrate
source and carbohydrate source plus BP supported the development of
H. macroceratitis protocorms with copious numbers of rhizoids. Stewart and
Kane (2006) reported a similar regulation of rhizoid production in H. macroceratitis where asymbiotic protocorms incubated in complete darkness produced numerous rhizoids while protocorms incubated in either a 16/8 h or
24/0 h L/D photoperiod produced either no or greatly reduced rhizoids.
Similar photoperiodic control of rhizoid production during asymbiotic orchid seed germination has been reported by Kauth et al. (2006) for the
orchid Calopogon tuberosus, while Arditti et al. (1981) reported that rhizoid
production was not controlled by photoperiod in species of Platanthera and
Piperia, related genera to Habenaria. The current data suggest that carbohydrate availability, not exclusively photoperiod, may play a role in the
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S. L. Stewart and M. E. Kane
production of rhizoids in orchid protocorms, which in turn contributes
to root development in more advanced seedling developmental stages.
Asymbiotic orchid seed germination media are typically supplemented
with a simple carbohydrate source (sucrose). Our current data support the
notion that most orchids can readily use a wide range of carbohydrate sources
when cultured in vitro, and that often difficult-to-germinate terrestrial orchids may follow this same trend. While orchid seed germinate and develop
readily on these simple carbohydrates, free simple carbohydrates, such as
sucrose, can be considered ecologically unimportant since carbohydrates
rarely exist in this form in nature (Harley, 1969). Germinating seed and
developing plants in the wild are dependent upon their mycobionts for the
breakdown of complex carbohydrates (cellulose, starch) and transport of
the resulting simpler compounds (trehalose, mannitol) into plant tissues
(Harley, 1969; Smith, 1966, 1967). Future studies on the effects of simple
and complex carbohydrates on the germination of orchid seed and early
protocorm/seedling development should be conducted under both asymbiotic and symbiotic culture conditions to gain a more ecologically-complete
understanding of orchid seed germination physiology. Nonetheless, given
the prevalence of asymbiotic orchid seed germination as a tool in the commercial production of orchid plants and as a primary research tool, these
current data represent new information concerning the wide suitability of
simple carbohydrates during in vitro asymbiotic germination and early protocorm development.
ACKNOWLEDGMENTS
Funding for this work was provided by the Florida Division of Forestry
and the San Diego County Orchid Society. We thank Nancy Philman (University of Florida) for assistance in experimental design. Brand names are
provided for reference only; the authors do not solely endorse these particular products.
REFERENCES
Arditti, J. 1979. Aspects of the physiology of orchids. Advances in Botanical Research 7: 421–655.
Arditti, J., J. D. Michaud, and A. P. Oliva. 1981. Seed germination of North American orchids I, native
California and related species of Calypso, Epipactis, Goodyera, Piperia, and Platanthera. Botanical Gazette
142: 442–453.
Ballion, G., and M. Ballion. 1924. The non-symbiotic germination of orchid seed in Belgium. Orchid
Review 32: 305–309.
Clement, E. 1924. Germination of Odontoglossum and other seed without fungal aid. Orchid Review 32:
233–238.
Clement, E. 1926. The non-symbiotic and symbiotic germination of orchid seeds. Orchid Review 34:
165–169.
de Pauw, M. A., W. R. Remphrey, and C. E. Palmer. 1995. The cytokinin preference for in vitro germination
and protocorm growth of Cypripedium candidum. Annals of Botany 75: 267–275.
Downloaded by [University of Florida] at 13:59 22 March 2012
Carbohydrate Effects on Orchid Seed Germination
1165
Dutra, D., T. R. Johnson, P. J. Kauth, S. L. Stewart, M. E. Kane, and L. Richardson. 2008. Asymbiotic
seed germination, in vitro seedling development, and greenhouse acclimatization of the threatened
terrestrial orchid Bletia purpurea. Plant, Cell Tissue and Organ Culture 94: 11–21.
Ernst, R., and J. Arditti. 1990. Carbohydrate physiology of orchid seedlings III: Hydrolysis of maltooligosaccharides by Phalaenopsis (Orchidaceae) seedlings. American Journal of Botany 77: 188–195.
Ernst, R., J. Arditti, and P. L. Healey. 1971. Carbohydrate physiology of orchid seedlings II: Hydrolysis
and effects of oligosaccharides. American Journal of Botany 58: 827–835.
Harley, J. L. 1969. The Biology of Mycorrhiza. London: Leonard Hill.
Harrison, C. R., and J. Arditti. 1978. Physiological changes during the germination of Cattleya aurantiaca
(Orchidaceae). Botanical Gazette 139: 180–189.
Hew, C. S., and T. C. Mah. 1989. Sugar uptake and invertase activity in Dendrobium tissues. New Phytologist
111: 167–171.
Johnson, T. R., and M. E. Kane. 2007. Asymbiotic germination of ornamental Vanda: In vitro germination
and development of three hybrids. Plant Cell, Tissue and Organ Culture 91: 251–261.
Johnson, T. R., S. L. Stewart, D. Dutra, M. E. Kane, and L. Richardson. 2007. Asymbiotic and symbiotic
seed germination of Eulophia alta (Orchidaceae)—Preliminary evidence for the symbiotic culture
advantage. Plant Cell, Tissue and Organ Culture 90: 313–323.
Kauth, P. J., D. Dutra, T. R. Johnson, S. L. Stewart, M. E. Kane, and W. Vendrame. 2008. Techniques and
application of in vitro orchid seed germination. In: Floriculture, Ornamental and Plant Biotechnology:
Advances and Topical Issues, Volume V, ed. T. da Silva, pp. 375–391. Middlesex, UK: Global Science
Books.
Kauth, P. J., W. A. Vendrame, and M. E. Kane. 2006. In vitro seed culture and seedling development of
Calopogon tuberosus. Plant Cell, Tissue and Organ Culture 85: 91–102.
Knudson, L. 1916. Influence of certain carbohydrates on green plants. Cornell Agricultural Experiment
Station Memoirs 9: 1–75.
Knudson, L. 1922. Non-symbiotic germination of orchid seeds. Botanical Gazette 73: 1–25.
Knudson, L. 1924. Further observations on non-symbiotic germination of orchid seeds. Botanical Gazette
77: 212–220.
La Garde, R. V. 1929. Non-symbiotic germination of orchids. Annals of the Missouri Botanical Garden 16:
499–514.
Miyoshi, K., and M. Mii. 1998. Stimulatory effects of sodium and calcium hypochlorite, pre-chilling and
cytokinins on the germination of Cypripedium macranthos seed in vitro. Physiologia Plantarum 102:
481–486.
Rasmussen, H. N. 1995. Terrestrial Orchids: From Seed to Mycotrophic Plant. Cambridge: Cambridge University
Press.
Smith, F. E. V. 1932. Raising orchid seedlings asymbiotically under tropical conditions. Garden Chronicle
91: 9–11.
Smith, S. E. 1966. Physiology and ecology of orchid mycorrhizal fungi with reference to seedling nutrition.
New Phytologist 65: 488–499.
Smith, S. E. 1967. Carbohydrate translocation in orchid mycorrhizas. New Phytologist 66: 371–378.
Smith, S. E. 1973. Asymbiotic germination of orchid seeds on carbohydrates of fungal origin. New
Phytologist 72: 497–499.
Spoerl, E., and J. T. Curtis. 1948. Studies of the nitrogen nutrition of orchid embryos III, amino acid
nitrogen. American Orchid Society Bulletin 17: 307–312.
Stenberg, M. L., and M. E. Kane. 1998. In vitro seed germination and greenhouse cultivation of Encyclia
boothiana var. erythronioides, an endangered Florida orchid. Lindleyana 13: 101–112.
Stewart, S. L., and M. E. Kane. 2006. Asymbiotic seed germination and in vitro seedling development of
Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell, Tissue and Organ
Culture 86: 147–158.
Wynd, F. L. 1933. Sources of carbohydrate for germination and growth of orchid seedlings. Annals of the
Missouri Botanical Garden 20: 569–581.