Plant Cell Tiss Organ Cult (2008) 93:223–230
DOI 10.1007/s11240-008-9365-1
ORIGINAL PAPER
A classroom exercise in hand pollination and in vitro
asymbiotic orchid seed germination
Philip J. Kauth Æ Timothy R. Johnson Æ
Scott L. Stewart Æ Michael E. Kane
Received: 20 December 2007 / Accepted: 7 March 2008 / Published online: 26 March 2008
Ó Springer Science+Business Media B.V. 2008
Abstract While many scientific reports on orchid
seed germination provide germination protocols, few
provide concise descriptions of plant selection, hand
pollination, and asymbiotic seed culture for use in
classroom exercises. Another major limitation for
conducting orchid seed germination exercises is the
availability of seeds or flowers to pollinate. In this
paper, we outline an efficient and reliable classroom
exercise using the orchid Spathoglottis to demonstrate hand pollination and subsequent asymbiotic
seed germination. Flowers of S. parsonii are hand
pollinated and subsequent seed capsule development
is carefully monitored. Hand pollination of the orchid
flower provides an opportunity to discuss floral
morphology and associated reproductive biology.
Capsules are harvested 30 to 40 days after pollination, prior to capsule dehiscence. Spathoglottis
kimballiana seed capsules are surface sterilized,
seeds excised, and then sown on P723 Orchid Seed
Sowing Medium. Germination occurs quickly and
large seedlings ready for greenhouse acclimatization
develop within 4–6 months.
P. J. Kauth (&) T. R. Johnson M. E. Kane
Department of Environmental Horticulture, University
of Florida, P.O. Box 110675, Gainesville, FL 32611, USA
e-mail: pkauth@ufl.edu
S. L. Stewart
PhytoTechnology Laboratories, 14335 West 97th Terrace,
Lenexa, KS 66215, USA
Keywords Classroom laboratory Teaching
exercise Orchid pollination Orchid seed culture
Introduction
The rising popularity of orchids has created a demand
for high quality plants as well as industry workers
trained in orchid hybridization and in vitro asymbiotic
seed germination techniques. Asymbiotic techniques
involve germinating orchid seeds in vitro on a defined
medium containing carbohydrates, vitamins, minerals,
and a solidifying agent. Since orchid seed germination
requires training and practice, high school and college
instructors have expressed interest in learning these
techniques to incorporate them into science class
curricula. In addition, instructors and students can use
these methods to study the unique biology of orchid
flowers and seeds.
Many publications on asymbiotic germination
describe efficient methods; however, these methodologies are limited as teaching tools because clear
descriptions of hand pollination methods for generating seed capsules are often absent. Lo et al. (2004)
provided a protocol for pollinating orchid flowers and
subsequent seed germination, but the pollination
procedures are not descriptive enough for teaching
methods. Mudge and Chu (1992) provided a classroom exercise for orchid seed germination, but hand
pollination techniques are not provided. Hand pollination is critical to instructors since a reliable seed
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source is required to incorporate orchid seed germination into classroom exercises.
This paper presents a complete methodology to
incorporate asymbiotic seed germination in the
classroom, from flower pollination through subsequent seed germination and development. After
completing this exercise students will be able to:
(1) describe orchid flower morphology and associated
reproductive biology (2) recognize seed capsule
development and maturation (3) produce seedlings
using asymbiotic seed culture procedures.
Background instructional information
In nature, orchid seeds utilize mycorrhizal fungi
during germination as sources of carbohydrates,
nutrients, and water (Stewart and Kane 2007).
In vitro symbiotic germination, which has been
Fig. 1 Flower morphology
of Spathoglottis parsonii.
(a) Components of a flower;
scale bar = 1 cm. (b)
Location of the anther cap;
scale bar = 1cm. (c)
Gynostemium with pollinia
and anther cap; scale
bar = 1 mm. (d) Pollinia
with individual pollinium;
scale bar = 1 mm (e)
Ventral view of
gynostemium with anther
cap and pollinia removed;
scale bar = 1 mm (f)
Gynostemium with pollinia
attached after hand
pollination; scale
bar = 1 mm
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Plant Cell Tiss Organ Cult (2008) 93:223–230
studied since the late 1800s, is the co-culture of
orchid seeds and their symbiotic fungi. Lewis Knudson discovered asymbiotic germination in the 1920s
(Arditti 1967), and this is the preferred method of
producing orchids for commercial purposes.
Orchid flowers are trimerous, possessing three
petals and three sepals (Fig. 1a). The third petal is
modified into a labellum or lip typically found
oriented toward the bottom of the flower. Potential
pollinators use the labellum as a landing platform,
directing them to the gynostemium. A distinguishing
feature of the Orchidaceae is the gynostemium or
column, which is the fusion of the style, stigma, and
stamens (Dressler 1981). The anther cap and pollinia
are located at the front of the gynostemium, while the
stigma is located directly behind the anther on the
underside of the gynostemium (Fig. 1b, c). The
pollen grains are joined together into masses called
pollinia (Fig. 1d). During a pollination event,
Plant Cell Tiss Organ Cult (2008) 93:223–230
pollinators deposit pollinia onto the stigmatic surface
(Fig. 1e, f).
A successful pollination event depends on pollen
and flower age. Spathoglottis flowers remain open for
several weeks, while inflorescences continually
flower for several months (pers. observation). Pollinating young, fully open flowers is recommended
since pollen is most receptive 1–8 days after flowers
are open (see Proctor 1998; Shiau et al. 2002; Lo
et al. 2004 for data on other species). Likewise, using
young flowers less than one week from opening
ensures that the stigmatic surface is receptive to
pollen. After two weeks, flowers close and pollen
becomes brown and unreceptive (pers. observation).
Procedures
Plant material
Spathoglottis parsonii and S. kimballiana are used for
this exercise, but these techniques can be used for any
orchid. Spathoglottis orchids are easy-to-cultivate
tropical terrestrial orchids, and are reliable sources of
seed capsules for lab exercises. These orchids are
available at local plant/orchid nurseries and garden
centers. They grow well in a standard soilless potting
mix such as Fafard No. 2 (Conrad Fafard, Inc.,
Agawam, MA) or a mix containing a 4:1:1 v/v/v ratio
of organic compost (peat), sand, and perlite (Beltrame 2006). The orchids should be grown in 30–70%
shade under a natural photoperiod with a daytime
temperature between 20–26°C. Plants should be
fertilized bi-weekly with either a balanced fertilizer
at 150 ppm nitrogen or a slow release fertilizer.
Spathoglottis grow rapidly, and single shoots can
become large specimens within six months. Plants
may need to be divided and repotted on a regular
basis.
Spathoglottis have several attributes that allow for
efficient classroom demonstrations. First, plants
flower year-round under a natural photoperiod since
no specific cultural requirements are necessary to
induce flowering. Second, the column, anther cap,
and pollinia are noticeable without a microscope.
Third, seed capsule formation is nearly 100% after
pollination. Fourth, Spathoglottis are receptive to
both self- and cross-pollination. Finally, capsules
mature in 30–60 days compared to 75–120 days for
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Phalaenopsis capsules and 150–195 days for Vanda
capsules (Arditti et al., 1982).
Hand pollination procedure
Identify the column and anther cap on a fully opened
flower (Fig. 2a). Gently remove the anther cap and
pollinia with a tooth pick (Fig. 2b). Dislodge the
pollinia and anther cap from the gynostemium by
applying slight upward pressure to the bottom of the
anther cap (Fig. 2c). The pollinia will adhere to the
toothpick on contact. Carefully remove the anther cap
from the pollinia (if still attached) without dislodging
the pollinia (Fig. 2d). After removal, transfer the
pollinia to the same flower or another flower by gently
placing the pollinia onto the stigmatic surface (Fig. 2e,
f). Remove the pollinia from another flower before
cross-pollinating with the pollinia from the donor
flower. During this step, apply gentle upward pressure
against the stigma while retracting the toothpick to
maintain contact between the pollinia and stigma.
Seed capsule development and maturation time is
species, hybrid, and growing condition dependent. For
first time hand pollination, closely monitor flower
senescence, capsule development, and capsule dehiscence. No physical signs are visible indicating
Spathoglottis capsule maturity, but other species’
capsules may turn light green, yellow, or brown. Once
capsule development time is estimated by allowing
capsules to dehisce, repeat the hand pollination procedures. Harvest the capsule one to two weeks before
capsule maturity by cutting the capsule from the
inflorescence (Fig. 2g). Harvesting the capsule at this
point will ensure that the capsule does not dehisce
(Fig. 2h). To limit surface contamination during
harvest, handle capsules with forceps and use a clean
razor blade to cut the pedicel. Place harvested capsules
in a clean paper bag and store at 4–10°C for a maximum
two days, since capsules may dehisce soon after
collecting. Do not store capsules in the freezer or in
plastic bags. Freezing will cause permanent damage to
the capsules, and plastic bags promote fungal and
bacterial growth by limiting air exchange.
Medium preparation
Orchid seed germination media are available from
companies such as PhytoTechnology Laboratories,
LLC (Shawnee Mission, KS, USA) and Sigma-
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Plant Cell Tiss Organ Cult (2008) 93:223–230
Fig. 2 Spathoglottis
parsonii flower morphology
and hand pollination
sequence. (a) Flower
profile. (b) Location of the
anther cap and pollinia. (c)
Removal of the anther cap
with the pollinia. (d)
Removal of the anther cap
from the pollinia. (e)
Transfer of pollinia onto the
stigmatic surface. (f)
Gynostemium with attached
pollinia. (g) Green capsule
ready to harvest. (h)
Dehisced capsule. Scale
bars = 1.5 cm
Aldrich (St. Louis, MO, USA). Many commercially
prepared media are complete, requiring only the
addition of water before autoclaving and dispensing.
Media such as P723 Orchid Seed Sowing Medium,
Knudson C, and Vacin and Went are suitable for
Spathoglottis seeds. Media can also be prepared in
the laboratory or classroom by mixing and storing
concentrated stock solutions (see Arditti and Ernst
1993 for methods on preparing various media from
stock solutions).
Prepare one liter of P723 medium by adding 32.74 g
of powder to 1000 ml of distilled water in a 2-liter
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Erlenmeyer flask. Adjust the medium pH to 5.7 with
0.1 N NaOH and 0.1 N HCl. Autoclave one liter of
medium for 40 min at 117 kPa and 121°C, and allow to
cool for 30 min after autoclaving. Dispense 25–30 ml
of medium into 9 cm Petri dishes; one liter of medium
makes 30–40 Petri dishes. If an autoclave is not
available, a pressure cooker is an excellent alternative
to sterilize equipment and medium (see Bergman
2006). Alternatively, adding Plant Preservative Mixture (PPM) into germination media can suppress
bacterial or fungal contaminants temporarily (Plant
Cell Technology, Washington, DC; www.ppm4plant-
Plant Cell Tiss Organ Cult (2008) 93:223–230
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Fig. 3 Seed germination
procedure. (a) Addition of
bleach to a wash bottle for
capsule disinfection. (b)
Shaker table for agitating
wash bottles during
disinfection. (c) Decanting
of liquid from a wash bottle.
(d) Removal of the pedicel
from the capsule after
disinfection. (e) Removal of
remaining floral tissue from
the sterilized capsule. (f)
Bisection of the capsule. (g)
Bisected capsule with
immature seeds exposed
and removed. (h) Inoculated
Petri dish
tc.com). Microwaving germination media may be
suitable as well (see PhytoTechnology Laboratories
Orchid Media Selection & Use Guide). However, using
PPM or a microwave to sterilize media are questionable compared to an autoclave or a pressure cooker.
Seed culture procedures
In preparation for capsule surface disinfection, prepare
graduated cylinders, wash bottles, and 500–1000 ml
distilled water per student. Sterilize the graduated
cylinders and wash bottles for at least 10 min and the
water for 40 min at 117 kPa and 121°C.
Remove any dried flower parts from the capsule
prior to surface disinfection. Remove surface debris
by placing the seed capsule in a container covered
with cheesecloth secured with a rubber band. Rinse
the capsule under cool tap water for 10–15 min.
While the capsule is rinsing, prepare the disinfection
solution by mixing 50 ml sterile water and 50 ml of
bleach (6% sodium hypochlorite) in a sterile graduated cylinder. To facilitate surface disinfection, add
one or two drops of Tween 20 or another surfactant,
such as liquid dish soap, to the disinfecting solution.
After rinsing, place the seed capsule in a sterile
wash bottle, add 70% ethanol, and agitate the capsule
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for 30 s. Decant the ethanol by slightly loosening the
cover allowing the ethanol to slowly drain into a
waste container. After the ethanol wash pour enough
disinfecting solution into the wash bottle to cover the
capsule (Fig. 3a). Cover the wash bottle and agitate
for 10 min (Fig. 3b). If using a shaker table, agitate
the bottle at 100 rpm (Fig. 3b). After 10 min, check
the capsule. The capsule may have yellow or white
bleach marks, but should still be firm. If the capsule is
still dark green, agitate in the disinfection solution for
five additional minutes. After the disinfecting process, decant the solution (Fig. 3c). Add sterile water
to the container, rinse the capsule for 2 min, and
repeat two more times.
Once the capsule is rinsed, place it in a sterile Petri
dish under a laminar flow hood. If a laminar flow hood
is not available, a sterile transfer box can be constructed by cutting an opening in one side of a large
Fig. 4 In vitro germination
and development of
Spathoglottis seeds. (a)
Two week old cultures;
scale bar = 1 mm. (b)
Protocorms with leaves
after four weeks in vitro
culture; scale bar = 1 mm.
(c) Seedlings transferred to
larger culture vessels six
months after initial seed
inoculation; scale
bar = 1 cm. (d)
Acclimatized seedling six
months after sowing; scale
bar = 1 cm. (e) One year
old plant; scale
bar = 10 cm
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Plant Cell Tiss Organ Cult (2008) 93:223–230
clear plastic storage container. Disinfect the inside
surface of the container with a 10% bleach solution and
allow to dry. Hold the capsule with a sterile forceps and
remove the pedicel and any remaining floral tissue with
a sterile scalpel (Fig. 3d, e). Cut the capsule longitudinally with a sterile scalpel blade (Fig. 3f). Use a
sterile spatula or inoculating loop to remove and
dispense seeds from the capsule onto a Petri dish
containing the germination medium (Fig. 3g, h). Do
not place too many seeds on the surface of the
germination media, since high seed densities inhibit
germination and development (Rasmussen 1989). Seal
the culture vessels with one layer of sealing film such as
Parafilm (Pechiney Plastic Packaging Inc, Chicago, IL,
USA) or Nescofilm (Karlan Research Products, Santa
Rosa, CA, USA).
Place the seed cultures under cool-white fluorescent lights in a 16-hour photoperiod at room
Plant Cell Tiss Organ Cult (2008) 93:223–230
temperature (23–25°C). Check cultures periodically
and discard contaminated cultures. Germination
should occur within two weeks of seed inoculation
evident by green protocorms (Fig. 4a). Leaves form
on the protocorms after four weeks culture, and
seedlings develop within four months of seed sowing
(Fig. 4b, c). At this time, aseptically transfer seedlings to a larger culture vessel containing 80–100 ml
P723 such as a MagentaÒ GA7 culture box (Magenta
Corp., Chicago, IL, USA) or PhytoTech Culture
BoxTM (PhytoTechnology Laboratories, LLC).
After two to four additional months, seedlings are
ready for greenhouse acclimatization (Fig. 4d).
Remove seedlings from culture vessels and wash
the culture medium from the roots. Place seedlings in
a soilless potting mix. Place clear plastic humidity
domes or plastic bags over the seedlings for two
weeks, and gradually remove them over an additional
two weeks. Water seedlings when the top portion of
the potting mix becomes dry. Begin fertilizing
seedlings after removing the humidity dome. Seedlings grow quickly under greenhouse conditions, and
develop into medium size plants within 1 year that
may flower (Fig. 4e; for complete timeline see
Fig. 5)
This exercise represents an excellent opportunity
to incorporate both technical and horticultural aspects
of orchid hybridization as well as in vitro seed
germination into the classroom. In the 6 years of
conducting this laboratory, students often remark that
this is one of their favorite exercises. Since growing
orchids from seeds to mature plant is lengthy,
conducting this experiment early in the semester is
recommended. Following through with this exercise
until seedlings become mature plants may be difficult
if the course is offered semesterly. Instructors can
prepare seed cultures during the previous semester to
provide students an opportunity to acclimatize seedlings. However, the emphasis is seed germination and
seedling development, not obtaining mature plants.
Providing a complete protocol for instructors is
necessary to determine the proper timeline for
conducting this laboratory. This exercise teaches
students diligence and patience, and rewards them
with orchids raised from seed. This paper provides a
detailed and easy to follow protocol for hand
pollination of orchid flowers and asymbiotic seed
germination.
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Fig. 5 Timeline of seed germination to adult plant
Acknowledgements Brand names are provided as references;
the authors do not solely recommend or endorse these products.
The authors thank Nancy Philman and Daniela Dutra
(Environmental Horticulture Dept., University of Florida) as
well as an anonymous reviewer for excellent comments.
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