Beruflich Dokumente
Kultur Dokumente
2
A GUIDEBOOK IN ORCHID
MICROPROPAGATION
Produced by
ISBN ________________
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OUTLINE OF TOPICS
I. Introduction on Orchid Growing (Lecture).
A. The value of orchid species and hybrids
B. Brief History of Orchid Embryo Culture
C. An introduction to Plant Tissue Culture
1. The Laboratory
2. The Culture Media
3. The Explant
4. The Techniques
III. Things needed for the Embryo Culture Technique (Visit the Lab).
A. Chemicals
B. Glasswares and Plastic wares
C. Instruments / tools
D. The Explant
V. Appendix
A. Formulation for the Knudson C Media
B. Formulation for the Vacin & Went Media
C. Formulation for the Murashige & Skoog's Media
D. Formulation for the Yamada's Media
E. Formulation for the R Media
F. List of Chemical, Equipment, Supplies & their suppliers
G. Orchid Pod Harvesting Schedule
VI. References
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Chapter I
INTRODUCTION TO ORCHIDS
Orchids are one of the most diverse and advance groups of plants on planet Earth. Its
size ranges from the tiny and miniscule botanicals to the gigantic forest epiphytes; with flowers of
various shapes, sizes color and scents, and inhabiting a wide degree of environment. Orchid
habitats are widely distributed and they are found in almost all continents except Antarctica.
The Philippine local folks have an extensive use for orchids, specially the genus
Dendrobium (Palmer, 2001). Dendrobium taurinum was used to make a wash to remedy loss of
hair. A tonic decoction was made from Dendrobium crumenatum and it was also used for ear
ache and ear infections. The old stalks from this orchid were cut and used as ties. The dried
stems of Dendrobium heterocarpum were used to make a belt to hold up the loincloth. The stems
or canes of several species of Dendrobium, including D. macrophyllum, have had local use in
various aspects of weaving, basketry and wickerwork. D. crumenatum is used is used for straw
plaiting and making straw hats. The yellow material used to decorate artifacts is provided by local
species of Dendrobium. This is due to the fact that the orchid stems turn yellow on drying, the
color being intensified by exposing the stems to heat from the sun or fire. The outer covering of
the stems are cut into strips, which are either woven into artifacts such as mats and armbands, or
fixed around objects such as arrows and fire sticks. The many types of articles decorated in this
way include domestic implements, clothing, body ornaments, cremonial articles, funerary relics
and weapons.
The yellow strips made from D. crumenatum stem were used for decorative purposes in
basketry and hat making, and D. heterocarpum was used to decorate items of clothing. D.
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tetraedre is used in small hand-woven baskets and in cigar cases. Yellow bark from D.
secundum is used to decorate bows and arrows, personal ornaments and funerary relics.
Various orchid clubs has also been created due to the hobby. Serious Filipino orchid
collectors bring into the country thousands of dollars worth of orchids, building large nurseries
and greenhouses just to house their valuable collections.
The Philippines is home of a diverse orchid flora which is composed of more than a
thousand species. However, only a few of them are cultivated as ornamentals or for cutflowers,
and some have become ancestors of the colorful orchid hybrids of today. Some of the species
are valued as botanicals and have inconspicuous short lived flowers. Some of the popular orchid
genera grown by hobbyists are Aerides, Arachnis, Ascocenda, Cattleya, Cymbidium,
Dendrobium, Doritis, Grammatophylum, Oncidium, Phalaenopsis, Paphiopedilum, Renanthera,
Spathoglottis, Trichoglottis, and Vanda. Some well know inter-generic hybrids are
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Brassolaeliacattleya, Kagawara and Mokara. With the ideal conducive climate the Philippines
has, both tropical and semi-temperate orchids could be grown in the country, making the
archipelago an ideal propagating area for orchids.
Orchid Propagation
In the early years of orchid culture, people have no idea of how to propagate these
plants. Orchid hobbyist usually get their orchids from forests. Then, they found out that orchids
also produce fruits (capsules) when their flowers are pollinated naturally by bees or artificially by
man. Each orchid fruit contain thousands of seeds.
Orchid seeds are very small (about 470-560 microns long, 80-129 microns wide) and
weight about 6 micrograms. Their size is one of the factors which gave them the tendency to be
disperse by wind for thousands of kilometers, away from their original habitat. However, not all of
these seeds germinate and grow into mature plants in nature. Most (but not all) orchid seeds
need a symbiotic fungus or mycorrhizae, usually of the genus Rhizoctonia, in order to germinate.
The fungus needs to infect the seeds in order for it to survive in its early stages of development.
The mycorrhizae infects the basal part of the seed and release an enzyme which converts starch
in the surrounding area into simple sugars. These will be the energy source or food of the
germinating embryo up to its development into a mature plant. Not all of the seeds grow in
nature. Only the seeds which land on a suitable surface (a rock, a bark of a branch or on the
ground) and infected with its particular mycorrhizae, germinates and grows into a plant. This
comprise only about an average of only 1%. However, through science and technology, almost
99% of the seeds could now be raised into mature plants through embryo culture.
Embryo culture or embryo rescue is one of the techniques used in the commercial
breeding and propagation of orchids. Thousands of plants could be produced in this method in a
year, due to the fact that orchids literally produce thousands to millions of seeds (about 6,200 in
Cephalanthera grandiflora and 2-3 million in Cattleya labiata). In this method the viable seeds or
ovules are sterilized and placed inside a flask containing artificial growing media. The media
consist of mineral salts, vitamins, amino acids, sugars and growth hormones. After a year or so
inside a flask, seedlings are then brought out into the nursery where they grow into maturity.
From these plants that are produced, a breeder usually selects the best flowering plant, registers
it and then clones this selected plant through the conventional division, cutting or kiekis method,
or better still, through plant tissue culture.
Plant tissue culture, particularly meristem culture or mericloning is the most efficient way
of mass producing a selected species or hybrid. In this method, the shoot tip or very young
inflorescence is severed, sterilized, and its actively growing region is obtained and cut into minute
pieces and inoculated into a flask containing artificial culture media. The tissue is permitted to
undergo callus formation, and then grows into minute orchid plantlets. These callus and plantlets
are then further divided to produce the required number of plants inside the laboratory. When the
right number of plants are obtained, then, the plantlets are then hardened and transferred into the
nursery where they are permitted to grow into maturity. The plants produced in these way are
true-to-type and identical to that of the mother plant.
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Chapter II
INTRODUCTION TO PLANT TISSUE CULTURE
Plant tissue culture is one of the biotechnological tool used in the mass propagation of
high value crops, specially orchids. Plant tissue culture is a broad term, which means the growing
or cultivation of plantlets or plant parts in an artificial culture medium under aseptic conditions. It
is a generic name which includes the following:
Note: the techniques below are all asexual means of propagation techniques.
b. shoot tip culture - This involves the culture of the apical meristem (part of the shoot tip)
attached to some leaf primordia in an artificial medium. The shoot tip tissue is much larger than
the one used for meristem culture. One disadvantage of this method is that it the breeder is
risking to actually kill the mother plant where the tissue was obtained.
c. meristem culture - This involves the culture of the apical meristematic dome only, which is
much smaller than that of the shoot tip tissue. However, the technique is similar to that of shoot
tip culture, but the leaf primordia are removed. This technique is also called mericloning, and the
one primarily used in mass propagation of selected hybrids and species. It is also effectively used
to produce virus-free planting materials.
d. tissue (or callus) culture - This involves the culture of tissue arising from explants of plant
organs like meristems, leaves, flower, flower stalks or buds. When the explants are placed inside
a flask with nutrient media, they are normally induced to undergo callus formation (unorganized
and undifferentiated masses of cells), wherein protocorm like bodies are formed. From this
tissues, will arise whole plantlets.
e. organ culture - This involves the culture of isolated plant organs like leaf, flower or inflorescent
and stem. The explant are usually excised, sterilized and inoculated into flasks with artificial
culture media. They usually do not undergo callus induction. It is similar in almost all aspect to
tissue culture, just that organ culture uses a much larger tissue -- an organ.
f. anther culture - This involves the culture of orchid anther (correctly called pollinia) or immature
pollen grains in an effort to obtain a haploid cell or callus line. However, orchids callus usually
automatically double in chromosome number, and literally produce diploid or sometime polyploid
cell lines. From these cells or callus, complete plantlets arise. The stage of pollinia development
is an important aspect in the success of this propagation technique. An application of these
technique is the production of orchids with recessive traits e.g. albinism.
g. cell suspension culture - These involves the culture of isolated cells or very small aggregates
of cells remaining dispersed in liquid medium. The cells usually comes either in organ or tissue
culture technique. From these cells, new plantlets could be regenerated. This technique could
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also be used in cell fusion, creation of genetically modified orchids (e.g. bombardment with gold
particles coated with foreign DNA), or production of secondary metabolites (like scents or
pigments).
h. protoplast culture - This involves the culture of naked cells (plants cells devoid of their cell
walls); which is a prerequisite for cell fusion, DNA insertion or for counting chromosomes
(karyotyping). The cells usually comes from a cell suspension culture, and could also
regenerated back into complete whole plants.
2. Disease Elimination - recover plants from pathogen (usually of viral diseases, but also to
recover from bacterial and fungal diseases); thus, what is produced are disease free plantlets.
Primary technique used is meristem culture.
3. Embryo Rescue - artificial culture of immature seeds (ovules); as long as pollen tube has
fertilized egg. There are many advantages:
a. For shorter breeding cycle --> e.g. dendrobium orchids 4-6 months for capsule to
mature, but 3 month old (about 75% of the waiting time) capsules can be used for ovule culture.
b. To prevent abortion of interspecific or intergeneric crosses
5. Production of Polyploid Plants - through the use of colchicine or other chemicals which
could induce chromosome doubling; polyploid orchids are produced. Colchicine is usually
incorporated into the culture media where the seeds will be grown. Polyploid orchids are known
to have a much thicker petal and larger flowers.
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8. Genetic Engineering - this involves the formation of new combination of genetic material.
Done through insertion of foreign DNA into target cell, use of genetic bombardment gun with gold
particles coated with DNA; Other vectors include Agrobacterium, DNA or RNA virus, T plasmid or
pure DNA (direct gene transfer through DNA uptake, microinsertion or electroporation. Then
target cell permitted to develop into whole organism which will express the gene. Gene usually
with anti-biotic resistance markers.
10. Use in Basic Research - for better control of the factors affecting growth: minimize
correlation. This is to improve techniques in micropropagation.
b. Physiological Studies
(1) Metabolism - cell cycle, respiration, DNA & RNA, photosynthesis
(2) Nutrition - deficiency or toxic levels of nutrients
(3) Effect of plant growth regulators
11. Convenience in Transporting Plants -- tissue cultured orchids still in flask are easier to
transport, less bulky and will not likely be subjected to quarantine.
ADVANTAGES OF MICROPROPAGATION
1. Produces numerous propagules in relatively short period of time
2. Uses relatively smaller space than conventional propagation methods
3. Propagation can be done all year round independent of seasonal changes
4. Produces large number of disease-free planting materials, free from viruses, fungus, bacteria
5. Produces clones of plants that are slow and difficult or impossible to propagate vegetatively
6. Long term conservation or storage of vegetatively propagated plants; free from environmental
risks
7. Propagules are less bulky to transport, and not subject to quarantine
8. No labor and materials for watering, weeding and spraying of pesticides while inside flasks.
9. No care or attention needed between subculture
DISADVANTAGES OF MICROPROPAGATION
1. Requires high skill for successful operation
2. Requires specialized and expensive production facility, laboratory and greenhouse
3. Fairly specific methods are necessary to obtain optimum results from each species
4. Labor intensive (due to subculture), resulting in high cost of propagules.
5. Technique may pose other problems like contamination, vitrification, non-compatibility with
media or inability to survive when transferred into greenhouse from laboratory.
6. Plantlets obtained are initially small, not autotrophic and susceptible to water loss --> undergo
a transition period of hardening in the greenhouse
7. Can produce genetically aberrant plants due to extensive use of plant growth regulators.
Therefore, to prevent this from happening, use only plants at 7th subculture; then replace "old
cultures" with fresh new initial cultures.
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CHAPTER III
DESIGN FOR AN ORCHID LABORATORY
Success in orchid plant tissue culture depends upon various factors like
available facilities, choice of nutrients, type of growth regulators, type of explant
used, time of explant collection, intensity, duration and type of light, temperature
and other variants. The most important factor is that all operations require
aseptic conditions, thus absolute cleanliness and orderliness must be
maintained throughout the laboratory. Sterilization techniques should be
meticulously followed to avoid any contamination of the samples. Design of the
tissue culture laboratory usually is specific and fitted with filtered air inlets and
decontamination facilities as well as temperature and humidity controls.
Because of the rapid advances in this field, there is an increasing need for
training and developing skills in the techniques required for modern plant
biotechnology and its applications.
In designing any laboratory, big or small, certain elements are essential for
a successful operation. The correct design of a laboratory will not only help
maintain asepsis, but it will also achieve a high standard of work.
FACILITIES
Careful planning is an important first step when considering the size and
location of a laboratory. It is recommended that visits be make to several other
facilities to view their arrangement and operation. A small lab should be set up
first until the proper techniques and markets are developed.
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Larger labs are frequently built as free-standing buildings. Although more
expensive to build, the added isolation form adjacent activities will keep the
laboratory cleaner.
4. The floor should be concrete or capable of carrying 50 pounds per square foot.
Walls and ceiling should be insulated to at least R-15 and be covered inside with
a water-resistant material.
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over air intakes to the laboratory or on furnaces. If possible, an enclosed
entrance should precede the laboratory; sticky mats can be laid there to help
collect dirt from the outside, or shoes can be removed.
Unusual requirements for electricity and fire safety dictate that power
installation be done by professional electricians. Most wiring will require 110
volts. Temperature and fire alarms are to be connected directly to telephone lines
to give fast warnings of problems. An emergency generator should be available
to operate essential equipment during power outages.
The glassware washing area should be located near the sterilization and
media preparation areas. When culture vessels are removed from the growth
area, they are often autoclaved to kill contaminants or to soften semi-solid media.
The vessels can be easily moved to the washing area if the autoclave or
pressure cooker is nearby. Locate the glassware storage area close to the wash
area to expedite storage; these areas also need to be accessible to the media
preparation area.
The glassware area should be equipped with at least one large sink; two
sinks are preferable. Adequate work space is required on both sides of the sink;
this space will be used for glassware soaking tubs and drainage trays. Plastic
netting can be placed on surfaces near the sink to reduce glassware breakage
and enhance water drainage. The pipes leading from the sink can be PVC to
resist damage from acids and alkalis. Both hot and cold water should be
available with water distillation and/or deionization devices nearby. Mobile drying
racks can be stored nearby and lined with cheesecloth to prevent water dripping
and loss of small objects. Locate ovens or hot air cabinets (75 C) close to the
glassware washing and storage area. Dust-proof cabinets, low enough to allow
easy access, can be used in the storage area.
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MEDIA PREPARATION AND STERILIZATION AREA
The water source and glassware storage area should be convenient to the
media preparation area. Benches, suitable for comfortable working while
standing (34 to 36in.) and deep enough (24 in.) to hold equipment listed below
are essential. Their tops should be made with molded plastic laminate surfaces
that can tolerate frequent cleanings.
4. Hot plate/stirrer--At least one hot plate with an automatic stirrer is needed to
make semi-solid media. This purchase can be eliminated by using a stove and
hand stirring the media while it heats; however, the time saved by using a stirring
hot plate is worth the money spent.
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water, glassware, and utensils. Certain spores from fungi and bacteria will only
be killed at a temperature of 121 F and 15 pounds per square inch (psi). Self
generating steam autoclaves are more dependable and faster to operate.
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placed over bulbs to direct their light. Heat generated by the lights may cause
condensation and temperature problems. In addition to using procedures
previously mentioned, small fans with or without polyethylene tubes attached,
can be placed at the ends of shelves to increase air flow and decrease heat
accumulation.
Shelving within primary growth rooms can vary depending upon the
situation and the plants grown. Wood is recommended for inexpensive, easy-to-
build shelves. The wood for shelves should be exterior particleboard or plywood
and should be painted white to reflect the room's light. Expanded metal is more
expensive than wood, but provides better air circulation; wire mesh of 1/4 or 1/2
in. hardware cloth can be used but tends to sag under load. Tempered glass is
sometimes used for shelves to increase light penetration, but it is more prone to
breaking. Air spaces, 2 to 4 in., between the lights and shelves will decrease
bottom heat on upper shelves and condensation in culture vessels. A room that
is 8 ft high will accommodate 5 shelves, each 18 in. apart, when the bottom shelf
is 4 in. off the floor. The top and bottom shelves may be difficult to work.
In addition to the primary growth room, the aseptic transfer area needs to
be as clean as possible. It is preferable to have a separate room for aseptic
transfer; this decreases spore circulation and allows personnel to leave shoes
outside the room. Special laboratory shoes and coats should be worn in this
area. Laminar flow hoods or still-air boxes can be placed in this room and used
for all aseptic work. Ultraviolet (UV) lights are sometimes installed in transfer
areas to disinfect the room; these lights should only be used when people and
plant material are not in the room. Safety switches can be installed to shut off the
UV lights when regular room lights are turned on. Surfaces inside the aseptic
transfer area should be smooth to minimize the amount of dust that settles.
Several electric outlets are to be installed to accommodate balances, flow hoods,
bacti-cinerators, and microscopes.
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LABORATORY SET-UP AND CHECKLIST
OF PTC REQUIREMENTS
1. Parts of a TC Laboratory
a. Preparation Room
b. Inoculating Room
c. Culture Room
d. Greenhouse/Nursery
3. Laboratory Furniture
a. Working Table g. Culture shelves
b. Filing Cabinet h. Drying Racks
c. Hood Table i. Push Carts
d. Book Shelves j. Chairs / Stools
e. Ladder k. Office Table
f. Cabinets l. Shelves
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5. Laboratory Utensils
Pipette Pump / aspirator Scalpel
Pippetor Forcep
Scooper Scraper
Knife Spatula
Scissors Microspatula
6. Laboratory Supplies
a. LPG Gas tank r. Rubber Gloves
b. Knitted Cotton Gloves s. pH Paper
c. Ethyl Alcohol t. Pitcher
d. Washing Sponge u. Rubbing alcohol
e. Soap Detergent v. Rubber stoppers
f. Cotton w.. Surgical Gauze
g. Surgical Blade x. Fire Extinguisher
h. Waste Basket y. Rubber Bands
i.Fluorescent Lamps z. Used Paper ii. Polypropylene plastic
j. Bottle Brush aa. Bottle caps jj. Water drum.
k. Slippers bb.Face mask kk. Plastic Trays
l. Mop Head cc. Cloth Rugs ll. Matches/Lighter
m. laboratory Gown dd. Funnel nn. Insecticidal spray
n. Hand sprayer ee. Marking Pens oo.Pot Holder
o. Plastic Cover ff.Ballast pp.Light Starter
p. Record Books gg. Pencils qq.Ballpen
q. Aluminum foil hh. wax paper
7. Media Supplements
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Chapter IV
ASEPSIS: STERILIZATION OF MATERIALS
Asepsis is a technique of sterilizing all materials used in plant tissue culture. A sterile
environment is one of the prerequisite of a successful micropropagation venture. Thus, this
technique is one of the most important thing a plant tissue culturist need to master.
Ideally, a culture vessel only contain one species of plant, and nothing else. Any other organism
included in the vessel is considered a contaminant. The plant tissue culture media contains a
high concentration of sucrose and support the growth of many microorganisms. Microorganisms
like bacteria and fungus, upon contact to the media, grow much faster than the plant material
and, in a very short period, will overpopulate the vessel, compete with the found source and will
finally kill the plant since.
Thus, it is important to eliminate contamination, so that only the selected plant species grows in
the culture container. However, contamination is sometimes difficult to control, since there are
many sources where they could enter. Contamination could be air-borne, water-borne or in the
outer surface or inner tissues of the explant. Even if the culture media is initially sterilized,
bacterial or fungal spores could unknowingly be included usually during inoculation or
subculturing. This happens when utensils, explants or the hands are not properly sterilized. Dirty
working environments could also be a problem. Tiny insects like ants or mites could also enter
small holes in the culture vessels, causing contamination outbreaks.
One of the most effective and efficient means of sterilizing the culture media is through
steam sterilization in an autoclave or a pressure cooker. The minimum time necessary for steam
sterilization of a given amount of media is given below (based on Biondi & Thorpe, 1981)
assuming it is subjected to 15 psi (1.06 kg/cm2) and 250 oC :
20 to 50 ml 15 minutes
75 to 225 ml 20 minutes
250 to 500 ml 25 minutes
1000 ml 30 minutes
1500 ml 35 minutes
2000 ml 40 minutes
The practice is to give more time to those with larger volume media in containers, than those in
the lesser volume ones. An example: if sterilizing testtubes with 10 ml media; mayonaise bottles
with 30 ml, distilled water in Erlenmeyer flasks at 100 ml and in large E flask with 500 ml media ;
priority is given to those with 500 ml media, and thus the whole batch is sterilized at the condition
of the larger container which is 500 ml --> sterilize at 25 minutes.
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MICROWAVE STERILIZATION (1991) Microwave sterilization is now done in some plant tissue
culture media. However, this technique is not very reliable due to uneven heating. In microwave
ovens, heat is caused by turning water molecules at 360 degrees turn, not directly to heat energy.
Thus, some regions of the media could not be properly sterilized.
FILTER STERILIZATION -- used to sterilize heat sensitive proteins, amino acids, growth
hormones, vitamins
NOTE: Use Mercuric chloride as a sterilant as a last resort. DO NOT dispose mercuric chloride
into the sink as it is very toxic. Instead, contain the used sterilant solution in a sealed plastic
vessel.
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CHAPTER V
BREEDING TECHNIQUES:
POLLINATION & POLLEN STORAGE
Orchid flowers need to be pollinated in order for it to form a fruit, which contain the seeds.
Orchid flowers are usually pollinated 4 days after the flowers have opened, however some
breeders has found out that orchid flowers could already be pollinate while still closed.
Orchid flowers are hermaphrodites, meaning each flower have a male pollinia and a
female stigma (see Parts of an Orchid Flower). Orchids are unique group of plants, having the
male and female flower parts fused into a column. Also, all orchids share the same floral
arrangement of having 3 sepals (1 dorsal, 2 lateral) and 3 petals (2 lateral, one modified into a lip
or labellum). The pollinia is a mass of pollen grains, usually covered by an anther cap. Pollination
is accomplished by placing the pollinia into the stigma of the flower. If the pollinia is placed into
the stigma of the same flower or of the same plant, this is called self-pollination. However, if the
pollinia is placed into the stigma of another orchid species/cultivar, then it is called cross-
pollination.
Once pollination is successful, the petals and sepals of the flower start to dry up, but the
ovary remains green and starts to enlarge. The ovary continues to enlarge until it forms into a
capsule, and after a few months, it is already mature and ready for embryo culture.
POLLINIA STORAGE
There are times when the selected female and male flowers will not bloom at the same
time. Thus, one option is to store the pollinia until the female flower is available. The procedure
of pollinia storage is described below:
1. Remove the anther cap and collect the pollinia from the intended male parent using a forcep or
toothpick.
2. Carefully remove translucent stalk (stipe and viscidium, like in Phalaenopsis flowers) holding
the pollinia to prevent possible microbial contamination.
3. Wrap the pollinia in clean dry tissue paper, place it in dry vial or test tube. Store in dessicator
and place it in dry shelf of the refrigerator. Dessicator salts can be silica gel (usually purchased in
drug stores along with your medicines in bottles) or calcium chloride (CaCl2) salts wrapped in
tissue paper and placed at bottom of bottle. A pinch of calcium hypochloride salts wrapped in
tissue paper may be added to prevent fungal growth inside the dessicator.
4. Label the test tube. Pollinia may be viable for a few months. Use the pollinia when the
selected female flower is available.
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POD HARVESTING
Pods are harvested once they are mature and or have burst. This means that the pollen
tube of the pollen have reached the ovules and have fertilized it. The grower has the option of
selecting the technique in which he could apply to sow his orchid seeds. Thus this would affect
the schedule of his pod harvesting.
There are two methods of sowing seeds in orchids. They are the dry pod culture and the
green pod culture. The dry pod culture is also called seed culture since the explant to be used
are the mature viable seeds. In this method, the orchid capsule have already burst, and the
seeds are ready to be carried by the wind. Seeds are powdery or dust-like, and are white, yellow,
or brown in color. It takes a long time for the orchid capsule to mature (4-10 months), and
usually, as it opens, some of the seeds are already lost. Thus, it is very important that orchid
capsules be checked regularly for signs of maturity (yellowing and presence of cracks). Once the
capsule is ready it is cut off from the flower stalk and wrapped in dry tissue paper and brought to
the laboratory. Seeds in the capsule have to be properly dried in a dessicator. A dessicator is
usually composed of a large bottle with lid and with a lining of metal screen at the bottom.
Beneath the metal screen, is the desiccant pockets which could be obtained from drug stores.
The desiccant pocket is composed of chemical salts (silica gel or calcium chloride) which absorb
moisture from the air inside the bottle. Once dried, the seed could also be stored for some time in
the refrigerator or immediately sown in flasks.
In green pod culture, also called embryo culture or embryo rescue, the immature seeds
are the once used as explant. In orchids, the interval from pollination to fertilization varies from
one genera to another and varies from 10 days to 6 months. Studies have shown that orchid
embryoes become viable and are capable of germination soon after fertilization. This takes place
long before fruit maturity. Green pod culture offers the advantage of shortening the time from
pollination to flasking. Below is a table showing the time (in days) when immature capsules from
the following genera are ready for flasking
Table 1. Number of days when seed pods of certain genera will be ready for green pod culture.
(Also depends on species and cultivar)
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CHAPTER VI
MEDIA PREPARATION AND STERILIZATION FOR
ORCHID EMBRYO CULTURE
2. In a 1000 ml beaker, dissolve the first chemical (a = ammonium sulfate) in approximately 900
ml distilled water.
3. Bring the volume to 1000 ml in a volumetric flask using distilled water.
4. Place and store the stock solution in a tightly covered brown bottle at room temperature.
5. Repeat the procedure for all the chemicals. Dissolve each chemical separately and place them
on separate brown bottles.
6. Label the bottles with the corresponding chemicals with the name, date prepared and amount
to be used per liter media.
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Note: Sodium EDTA is used as chelator for Ferrous sulfate, to prevent it from precipitating.
Ferrous sulfate will not likely to dissolve without sodium EDTA. Use this stock at 10 ml each per
liter of stock solution.
1. Dissolve 56.0 g KOH (or 40.0 g. NaOH pellets) in approximately 900 ml distilled water. This is
an exothermic reaction, wherein the solution will be a little bit warmer than before.
2. Cool the solution to room temperature.
3. Dilute or bring volume to 1 liter in a volumetric flask using distilled water.
4. Transfer to brown glass bottle and label.
NOTE: 1 N KOH is used when the nutrient medium has a low pH (pH below 5). Add 1 N KOH
drop by drop until the desired pH (5-6) is attained. Be careful with the said chemicals, they are
corrosive.
NOTE: 1 N HCl is used when the nutrient medium has a pH higher than pH of 5.6. 1 N HCl is
added drop by drop until the desired pH is attained. In pipetting out the pure HCl for dilution, do
this inside a fume hood. Be careful with the said chemicals, they are corrosive.
Vitamins usually enhances growth of cultures in vitro. Vitamins used in orchid embryo culture
includes Vitamin B complexes which help in the cells' metabolism. Vitamin B complex from drug
stores can also be used.
The vitamin stock solution below is that of Nitsch & Nitsch.
2. Add enough distilled water to make a 500 ml solution in a volumetric flask. Folic acid will not
usually dissolve.
3. Adjust pH to 7.00 to dissolve Folic acid by adding drops of 1 N KOH or NaOH. A clear
transparent solution will result.
4. Store vitamin stock in a brown bottle and inside the refrigerator.
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MYO-INOSITOL STOCK
This stock could replace coconut water. To make the stock solution, dissolve 5 grams of Myo-
inositol powder in enough distilled water to make a 500 ml solution. Use 10 ml of this stock in
making 1 liter media. Store the solution in a brown bottle and in a refrigerator.
Procedure:
1. Strain / Filter the coconut water (not coconut milk) using cheese cloth or cotton placed in a
plastic or glass funnel to remore floating impurities. Get 100 ml of this and place in a 1000 ml
beaker. Excess coconut water is stored by placing it in a plastic container and stored in freezer.
6. Place 3 medium size tomatoes in a blender/osterizer and add 50 ml distilled water. Osterize
the tomatoes until a puree is produced. Strain the seeds and add the content into the media.
7. Weigh the agar and place in into a separate container. It is mixed with about 100 ml distilled
water in a glass (Pyrex) beaker or a small sauce pan. The mixture is made to boil in a magnetic
hotplate stirrer or a stove until the agar is cooked. The agar mixture is then added to the media.
10. The media is then dispensed into catsup bottles or Erlenmeyer flasks.
11. Seal the bottles with metal or plastic caps. Wrap the caps with paper sheets by using rubber
bands.
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PREPARING THE KNUDSON C REFLASKING MEDIUM
To Prepare 1 Liter Knudson C Reflasking Media, the following are needed:
Procedure:
1. Get 100 ml of coconut water and place in a 1000 ml beaker.
6. Place 100 grams Bungulan Banana (about 3-4, depending on size) a blender/osterizer and
add 50 ml distilled water. Osterize the bananas a homogenate is produced. Add the contents
into the media.
7. Weigh the agar and place in into a separate container. It is mixed with about 100 ml distilled
water in a glass (Pyrex) beaker or a small sauce pan. The mixture is made to boil in a magnetic
hotplate stirrer or a stove until the agar is cooked. The agar mixture is then added to the media.
10. The pH is adjusted to 5.6 (adding HCl or KOH) using a pH paper or pH meter.
11. The media is then dispensed into catsup bottles or Erlenmeyer flasks.
12. Seal the bottles with metal or plastic caps. Wrap the caps with paper sheets by using rubber
bands.
26
CHAPTER VII
MEDIA PREPARATION AND STERILIZATION FOR
ORCHID TISSUE CULTURE
PROCEDURE FOR IN VITRO PROPAGATION OF DENDROBIUM
i. INITIAL CULTURE
Procedure:
5. Adjust pH = 5.6
6. The media is then dispensed into ketsup bottles or Erlenmeyer flasks. Bottles
are covered with cotton plugs or rubber stoppers.
7. Sterilize the bottles in a pressure cooker for 30 minutes at 15 psi and 121 oC.
4. Swab with 95% ethyl alcohol and rinse with sterile distilled water.
27
5. Inside the laminar flow hood or sterile chamber, sterilize the shoot with 1%
calcium hypochlorite or 20% bleach with Tween 20 for 30 minutes.
6. In a sterile petri dish, rinse three times with sterile distilled water.
1. Reflask initial culture in liquid medium every 3-4 weeks. Agitate in rotary
shaker.
3. At the stage where the protocorms are available in a large number (e.g. with
30-50 flask), some maybe set to be transferred to liquid media and others for
plantlet differentiation (depending on the targeted volume of production)
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PROCEDURE FOR IN VITRO PROPAGATION OF VANDA
Procedure:
6. Adjust pH = 5.6
7. The media is then dispensed into ketsup bottles or Erlenmeyer flasks. Bottles
are covered with cotton plugs or rubber stoppers.
8. Sterilize the bottles in a pressure cooker for 30 minutes at 15 psi and 121 oC.
1. Select and cut a young inflorescence with undifferentiated buds not more than
5 cm. Long
4. Inside the laminar flow hood or sterile chamber, sterilize the flower bud with
20-50% bleach with Tween 20 for 30 minutes.
29
5. Rinse three times with sterile distilled water.
1. Reflask initial culture in liquid medium every 3-4 weeks. Agitate in rotary
shaker.
3. At the stage where the protocorms are available in a large number (e.g. with
30-50 flask), some maybe set to be transferred to liquid media and others for
plantlet differentiation (depending on the targeted volume of production)
30
PROCEDURE FOR FLOWER STALK PROPAGATION PHALAENOPSIS
Procedure:
3. Add 20 g. sugar.
6. Adjust pH = 5.6
7. The media is then dispensed at 10 ml each in test tubes. They are then
covered with cotton plugs or rubber stoppers.
8. Sterilize the test tubes in a pressure cooker for 30 minutes at 15 psi & 121 oC.
3. Wipe the stalks with cotton wet with 95% ethyl alcohol 2-3 times
4. Cut the stalk into sections from 1 cm to 2 cm length. Each cutting must have a
node.
31
5. Soak the cuttings in 20% bleach with Tween 20 solution for 20
minutes.
8. Dip cuttings in 5% bleach solution for 10 minutes and dip in sterile distilled
water for 2 to 3 minutes.
9. Dip the portion of cuttings in a test tube with the culture medium.
3. At the stage where the protocorms are available in a large number (e.g. with
30-50 flask), some maybe set to be transferred to liquid media and others for
plantlet differentiation (depending on the targeted volume of production)
32
MICROPROPAGATION OF CATTLEYA AND ALLIES.
Treatment:
1. Wash the shoot in tap water and detergent. Dip the shoot in ethyl alcohol for
10 seconds. Mix in 10% bleach for 15 minutes. Rinse briefly in sterile distilled
water and dry on sterile petri dish.
4. Excise the Whole buds, severing just below the point of attachment. These
may be cultured, or dissected further to obtain meristem with one or two pairs of
leaf primordia.
5. Media: Grow buds or meristems in medium with 100 ml/l coconut water. Best
started in agitated liquid medium but agar solidified medium is often satisfactory
and required for stage III.
Light: continous light at 100-300 footcandles from cool white fluorescent lamps.
Temperature:: 26 oC
Discussion: Cattleyas multiply better in liquid than on agar, at least initially. The
theoretical reason is that the agitation inhibits polarity (orientation). Once polarity
is established, the cultures put out shoots and roots and mature. The initial
growth that is desired for multiplication is a mass of protocorms. As soon as this
mass grows to 1 cm it should be divided and put back into liquid or agar medium.
When the culture is on agar frequent division will help upset orientation and delay
plantlet formation..
33
MICROPROPAGATION OF CYMBIDIUM SPECIES & HYBRIDS.
Treatment: Remove outer leaves from 3 cm shoots. Dip in ethyl alcohol for 2
seconds then mix in 10% bleach for 15 minutes. Rinse in sterile distilled water.
Remove remaining leaves. Excise meristem consisting of apical dome, two leaf
primordia, and a cube of tissue, all less than 0.5 mm.
After 3-4 subcultures, transfer to media with agar (KC or Morel and Muller) for
shoot and root development.
Temperature: 22 oC
CHAPTER VIII
INOCULATION AND SUBCULTURING
Preparation of Laminar Flow Hood Before Inoculation:
1. Clean the laminar flow hood surface with 75% ethyl alcohol in cotton. For transfer
chamber/cabinet, the surface is sterilized by wiping it with 10% chlorox solution.
2. Get the culture bottles with fresh media and spray they with 75% ethyl alcohol and wipe them
dry with cotton. Place the culture bottles inside the laminar flow hood. Do the same to the
alcohol lamp, the beaker with 95% ethyl alcohol and other glasswares to be entered inside the
laminar flow.
4. Afterwards, turn off the UV light and bring in the washed dry pods, sterilized Petri dishes,
scalpel, and forcep.
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Green Pod Culture Technique:
1. Mature orchid pod is washed with detergent (e.g. Teepol) and running water. Inspect pod for
holes, insect damage or rotting.
2. Inside the transfer chamber of laminar flow, the pod, scalpel and forcep is sterilized by dipping
in 95 % ethyl alcohol and flaming at least 3 times over an alcohol lamp.
3. Hands are washed with soap and water, and then wiped with 70% isoprophyl alcohol. At this
stage, all manipulation is done inside the laminar flow.
4. In a sterile Petri dish, the pod is placed and cut crosswise or lengthwise (depending on size of
pod).
5. The ovules (white, yellow or light yellow-brown in color) are scrapped off inoculated into the
germinating media.
6. The bottles are labeled (date and cultivar/species). Accession numbers can be used.
7. The bottles are placed in lighted culture shelves. After one week, contaminated cultures are
removed and sterilized (decontaminated) with procedure similar to sterilizing fresh culture media.
8. After 3 weeks, viable ovules will show signs of germination (enlargement of ovules and
greening). After 2 months or when protocorms are 1 cm long, cultures are ready for reflasking.
2. Tap the pod over the paper to release the dust-like seeds.
4. Mix 1-2 grams sugar in 100 ml water. Then place the solution into the vial or test tube with
seeds and add a 1-2 drops Tween 40 or detergent (it serves as a surfactant). Cap and shake the
test tube until the seeds are submerged or fully wet. Let the seeds in the solution for 16-24
hours. This technique will germinate any bacterial or fungal spores in the seed and it will make
them susceptible to the sterilizing solution afterwards. Some Fungal/Bacterial spores are usually
resistant to the sterilizing solution.
5. After 24 hours, pour out/remove the sugar solution, and leave the seeds inside. Pour 10%
Chlorox solution plus 1-2 drops Tween 80 into the vial. Cap and shake vigorously and let it stay
there for 10 minutes. Let seeds settle down. (A centrifuge could be very helpful in settling seeds
down in the bottom of the test tube)
7. At the last washing, leave a little sterile distilled water, and pour the seed and water mixture
into the germinating media. 1 or half a drop of the solution with seeds is enough for each flask.
35
8. Cover the inoculated flask and label (orchid species/hybrid name and date). Place the flask
into culture shelves exposed to 16 hours light per day. (Some orchids like Paphiopedalum
requires darkness and cold temperature in order to germinate).
9. Observe for seed germination or contamination. Contaminated cultures must be removed and
sterilized to kill the bacterial/fungal contaminant before being washed.
2. Cut 2 inches X 2 inches filter paper squares (it need not be sterile). The fold the filter paper
squares in half lengthwise or cross wise.
3. Place a pinch (about 1/10 of a teaspoon or less) or orchid seeds in the middle of the filter paper
square.
4. Fold the filter paper square in half, enveloping the seeds. Fold the sides and top of the folded
filter paper square 2x to seal the enveloped seeds.
5. Fasten the two tips of the envelope using a stapler. Do not puncture a hole into the envelope,
just fasten the topmost right and left corner of the envelope with the staple wire.
6. Inside a laminar flow hood, place the filter paper envelope in a sterile petri dish. Pour the
sterilizing solution (20% solution of commercial bleach and water plus some drops of Tween 20)
in it. Pour enough to cover the filter paper envelope, about half the level of the petri dish. Slightly
agitate the petri dish to mix the solution with the envelope. Do this for 15 to 20 minutes. The
orchid seeds will now slightly change its color, as being bleached by the chlorox solution.
7. After 15 - 20 minutes, decant the sterilizing solution and wash the filter paper envelope in
sterile distilled water 3x in the same Petri dish. Use sterile forcep in moving the filter paper
envelope.
8. Allow the filter paper envelop with the seeds to dry up in the air flow inside the laminar flow
hood for about 4 hours. Let it stay in the petri dish and place a glass cover slightly open to allow
the sterile air from the laminar flow hood to enter the Petri dish.
9. When the filter paper envelope is completely dry, remove the staple wires to open the seal of
the envelope using a sterile forcep and scalpel. A sterile surgical scissors can also be used to cut
the paper open.
10. Carefully hold the filter paper envelop over an opened culture bottle, using a sterile forcep,
and gently tap the envelope to release the powder like-orchid seeds into the flask containing
orchid germinating media..
11. Cap the bottle and label. Place the bottle in a lighted shelf inside the culture room. The orchid
seeds will germinate after 3 weeks. Discard any contaminated cultures.
12. Subculture the orchid protocorms after 2 months into flasks containing fresh reflasking media.
36
Reflasking:
1. Culture bottles to be reflasked are sprayed with 70% ethyl alcohol and wiped dry with cotton.
2. The culture bottles are placed inside the transfer chamber or laminar flow, together with bottles
with fresh reflasking media.
3. Hands are washed with soap and water, and then wiped with 70% isoprophyl alcohol.
4. Forcep, scooper and scalpel are sterilized by dipping in 95% ethyl alcohol and flamed over an
alcohol lamp. They are then permitted to cool for 1 minute.
5. Culture bottles with protocorms are uncapped and the mouth of the bottle is flamed over the
alcohol lamp.
6. Using sterile scooper, protocorms are removed from the bottle and subcultured into bottle with
fresh media.
8. The bottles are placed in lighted culture shelves. After one week, contaminated cultures are
removed and sterilized.
9. Growth of the orchid protocorms into plantlets are checked regularly. When plantlets are about
three (3) centimeter tall, they are ready for acclimatization and compotting.
Chapter IX
ACCLIMATIZATION AND COMPOTTING
Acclimatization is an adjusting process wherein the plantlets grown in vitro are gradually exposed
to higher light intensity, usually diffused natural sunlight beside a glass window or in the nursery,
and lower humidity. The cultures will stay here for 1 month before they could be compotted. For
some, the decreasing amount of moisture in the medium as the water is gradually absorbed by
the plant will also help in the adjusting process. This process will “teach” or induce the plant to
photosynthesize and to synthesize a much thicker epidermis.
3. The plantlets are then dipped in a dilute solution of fungicide (either Dithane or Captan) and
with a few drops of Rooting Hormone (Root Booster, Hormex or others) for 3 minutes.
4. The plantlets are then sorted out base on size and placed on a sheet of old news paper to
dry.
37
5. Clay/plastic pots (3 inches in diameter) are filled with broken charcoal at the bottom and lined
with chopped tree fern roots. The charcoal and tree fern roots are previously sterilized by
boiling them in water for 30 minutes.
6. Then, the plantlets are arranged into the community pots, with their roots embedded in the
chopped tree fern and their leaves or stems upright. Their roots does not need to be inserted
too deep into the chopped tree fern. There should be about 15 – 25 plantlets (depending on
the size of the plant) per community pot. Enough chopped tree fern roots are added to cover
the roots of seedlings in the community pots.
7. For larger seedlings, 1 inch in diameter clay/plastic pots (size 1 or smaller) can be used.
Potting medium used are just chopped tree fern chips or pre-soaked and sterilized coconut
husk. Plantlets are arranged singly with their roots carefully pressed in between two tree
fern chips or coconut husks.
8. The pots are then sprayed with water and placed inside clear plastic bags and closed with
rubber bands. The plastic bags with compots are placed in a 50% shade area of the nursery.
9. After one week, the plastic bag is opened but not removed.
10. After another week, the compot are removed from the plastic bag and placed in a much
illuminated area (about 60% light) and watered (sprayed) regularly. A weak solution of
orchid foliar fertilizer is applied 2 weeks after, when new root tips have appeared. Bright light
is the key factor in the successful adjustment of the seedlings.
11. The compots are gradually trained to a semi-shaded (75% light) light intensity, depending on
the type of orchid. Vandas, Oncidiums, Cattleyas and Dendrobiums need more light
compared to Phalaenopsis.
12. Once the seedlings in compots are large enough, they can be transferred to single pots. For
seedlings in single pots, they can be transferred later on to larger sized pots when they have
outgrown their container.
13. Seedlings need to be watered everyday, sprayed with fungicide and fertilizer once a week
and need to be regularly inspected for occurrence of pest and diseases.
OTHER SUBSTITUTES
Due to the fact that the giant tree fern is endangered and also the manufacturing of charcoal is
restricted (especially those manufactured from forest trees), there is a need to look for new
alternative sources of planting media for orchid seedlings. Some of these are: )a) use of coconut
coir dust / fiber instead of tree fern chips; (b) use of charcoal manufactured from ipil-ipil or
kakawate; and (c) use of synthetic foam (used in making uratex beds) or styrofoam instead of
charcoal as a substitute.
There are many techniques in compotting orchids from flasks, thus, one has to adjust and adapt
the technique that will suit in your garden or nursery.
38
CHAPTER X
PROPAGATION AND OTHER STRATEGIES IN
ORCHID CONSERVATION
The Philippines has a very rich and diverse orchid flora, which is
composed of more than a thousand species distributed among the country's
more than 1,700 islands. Many of the orchid species are endemic and have
become parents of some of the beautiful and colorful orchid hybrids of today.
Some of the more familiar and noteworthy Philippine orchid species belong to the
genera Aerides, Amesiella, Arachnis, Ascocentrum, Bulbophyllum,
Cirrhopetalum, Dendrobium, Dendrochillum, Doritis, Epigeneium, Eria, Euanthe,
Flickingeria, Coelogyne, Grammatophylum, Kingidium, Liparis, Macropodanthus,
Paphiopedilum, Phaius, Phalaenopsis, Renanthera, Rhynchostylis, Spathoglottis,
Trichoglottis, Vanilla, and Vanda. Orchids have been a favorite houseplant for
Filipinos due to their beautiful flowers, its exoticness and mystery. Due to this,
Europeans in the 1700's searched through our forests. They have now living
specimens of almost all of our species. Most of our orchid species are sought
after my foreign orchid collectors and value them very much.
Vanda sanderiana var. immaculata, Vanda sanderiana, Vanda merrilli var rotorii,
Trichoglottis brachiata, Aerides lawrencea var. alba, Dendrobium taurinum var.
album, Phalaenopsis micholitzii, Phalaenopsis mariae, Paphiopedilum anitum,
and Dendrobium anosmum (Sanggumay puti)
The Philippine forest is a natural home for orchids. In any given place,
there could be about thousands to millions of orchids clinging high up in tree
branches, in shaded forest floors, in open grasslands, in large rocks near rivers
or the sea, or in limestone cliffs. In their natural habitat, orchids reproduce
successfully on their own, without human intervention. This due to the fact that
their natural pollinator is present and also with the help of a symbiotic fungus or
mycorrhiza which provide nourishment to the germinating seeds. Some orchid
species literally grow wild like weeds, wherein they overly populate some tree
branches together with some ferns and other epiphytes. Some are even widely
distributed (like some Cymbidium, Dendrobium, Dendrochilum, Flickingeria,
39
Phalaenopsis, Paphiopedilum, and Spathoglottis) that they are found all over the
country. Also the fact that orchids produce thousands to millions of seeds, thus,
they could successfully repopulate orchid collecting areas as long as the area is
not destroyed.
However, some of our orchid species have become threatened due to the
destruction of their natural habitats and the conversion of these forests into
agricultural, industrial or residential areas.
On the other hand, some orchid species are only found growing in certain
areas (e.g. Paphiopedilum anitum). They are found only in specific sites, very
hard to find, and are very difficult to cultivate them out of its habitat.
The rarity of some orchids and its high demand prompted the increase of
prices of some orchid plants. And because of these, more people are attracted to
conduct widespread and indiscriminate collection in the forest. Without a halt and
caution, in this widespread collection and destruction of its habitat, some orchid
species will certainly become threatened or extinct. Philippine orchids are
national treasures and it is the obligation of Filipinos to conserve them for future
generations.
40
Paphiopedilum spp., Peristeria elata, Phragmipedium spp., Renanthera
imschootiana & Vanda coerulea
The Orchid Species Group was set up in August 1984 following the 11th
World Orchid Conference. The group has more than 90 members worldwide. The
group is assigned to create a list and evaluate species which will be placed in the
Appendix I, II, and III of CITES.
One problem with CITES is how each member country interprets it.
Sometimes, the country implementing it would make much stricter laws that that
in CITES. Also, conservation laws of different countries still have many problems
and loopholes which when analyzed, actually does not protect orchids in the wild,
or sometimes are not practical in today's highly technological age. One example
is the law that prohibits collecting endangered or threatened orchid species from
the wild. If the site where the orchids (orchids growing in big trees or located near
river) are growing will be cleared off for agricultural development, construction of
highway, be flooded for dam building or threatened by flood or volcanic eruption,
then surely the that orchid is doomed. Sometimes, orchid in the wild are
threatened by introduced pests and diseases, or its pollinators are now absent
due to the use of pesticides, and these will surely affect their natural way of
reproduction. Another is the fact that orchids produced thousands or millions of
seeds, and by just reproducing them in the laboratory, could surely repopulate
the forest.
The Philippines must adopt its own version of orchid conservation plan
which is practical. Through orchid societies, like the Philippine Orchid Society,
information on orchid conservation could be disseminate to Filipinos. By
coordinating with government offices, protection of the orchids' natural habitat
could be done and plans for mass propagation started.
Identify which orchid are endangered, threatened, and which are not.
The Philippines has its own Orchid Specialist Group (OSG), which is based at
the Botany Division of the National Museum. The OSG is part of the Species
Survival Commission of the International Union for the Conservation of Nature
and Natural Resources (IUCN). The Orchid Conservation Network of the
Philippines, which is composed of a local group like the Philippine Orchid
Society, the Botany Section of the National Museum (Red List Group), Ferns &
Nature Society of the Philippines, the Philippine Horticultural Society, and other
orchid societies are tasked to create a Red List which is an accurate list of which
of the orchid species are endangered, threatened, and which are not. Once the
data is produced, the group could concentrate on which orchid species will be
prioritized for mass-propagation.
41
Mass Propagation of Orchid Species. Embryo culture is a powerful tool
in mass producing orchids. It is a fact that orchids produce thousands to millions
of dust-like seeds per plant. If enough orchid plants could be pollinated and
produce a capsule, the seeds could be grown in designated plant tissue culture
laboratories and could be later be used to re-stock the forest or the ones used for
trade. With this, the habitat where the orchid species are collected will not be
touched.. Priorities could be given to endangered and threatened species and
also to those highly demanded for trade. Knowledge with breeding and genetics
is a requirement in this in order to prevent producing weak and inferior plants.
Also, learn how to vegetatively divide your plants. Both private and government
sectors could be tapped for this.
In collecting specimens from the forests, do not get all the plants. As
much as possible, be responsible enough to get only the seedlings, and leave
enough matured plants that could survive and reproduce for the next generation.
Educate the amateur collectors to collect only for their own and to
follow a certain code of collecting plants. Amateur collectors need to be
trained not to collect indiscriminately, from the wild, and always follow the code of
orchid conservation ethics. Collect only a number of plants per each species
which you can take care off. As much as possible, collect only seedlings and
42
leave enough matured plants in the area for future generations. Do salvage
orchids from threatened areas and learn to cultivate them.
Recognize & Purchase only Plants that will live in your Locality. There are
cool-upland growing and lowland-warm growing orchids. Purchase and collect
only the plants that will grow well and flower in your place. Also, buy only plants
that are well-rooted, well-established, and free from pest and diseases.
Grow Your Plants Well. Resolve to give your plants the best possible
culture. Apply fertilizers and pesticides, and provide the necessary environment
for your plants. If you do not have success with a certain species, learn how
others succeed with it before obtaining another plant.
Share Your Plants. The best insurance for your rare species or clone is a
division of it in another's care. Be willing to share divisions, and trade with other
growers. Consider propagating seed from rare plants in your collection.
Networking through your local orchid organization is an ideal way to meet
interested participants. Excess plants could also be donated or sold to other
orchid enthusiasts. You could also trade or change pollinia, seeds, seedlings, or
matured plants with other growers here or around the world.
Plan for Emergencies. Many collections are lost when the owner or
caretaker, for various reasons, is unable to care for them. Plan for these events.
Leave written instructions on how to take care of your plants if leaving the plants
to someone. Indicate which plant is rare or important. Indicate where records of
43
your collection are stored. Designate a contact person (who grows orchids well)
for family members to contact in the event of your disability or death, and plan
ahead for how plants should be disbursed or disposed of, so that important
plants are not simply lost.
44
CHAPTER XI
ESTABLISHING AN ORCHID BUSINESS
Orchid growing is one of the most popular and rewarding hobby in the
world, due to the orchids’ elegance, beauty, exotic flower shapes, and varied
colors. It is also a multi-million dollar business around the world, with large
corporations solely devoted in the production of specific orchid hybrids used for
the cut-flower and flowering potted plant business. With this, countless orchid
clubs has been formed to cater the needs of various orchid hobbyists and
enthusiasts, the discovery of new species, the continuous development,
registration and judging of new hybrids, and technical/consultative support for the
business aspect. Plant propagation technique like micropropagation has greatly
revolutionized the way these plant group has been produced, and thus, has
helped in the development of the business.
1. Cut-flower trade -- the large and colorful orchid flower like the Cattleya,
Cymbidium, Dendrobium and Vanda are ideal for corsage, bridal bouquets,
arm band, in flower arrangements, and also for lei or garland.
4. For Business – with the wide demand of orchids locally and abroad, various
plant propagating nurseries were established to mass produce a regular
supply of flowering orchids – both species and hybrids – to people in the cities
for their orchid needs. With these, orchids are produced like in an assembly
line like factories, starting with the systematic breeding of orchids,
germination of the seeds in sterile laboratories, acclimatization and growing of
the seedlings to maturity in the nurseries, scheduled flower induction of
plants, and marketing. The orchid is a high value crop mass-produced to
cater the demand of various clientele.
45
3. Phalaenopsis & Doritis
4. Dendrobium
5. Cattleya group
6. Paphiopedilum
7. Spathoglottis
8. Grammatophyllum
9. Cymbidiums
10. Oncidiums
11. Orchid Species & Botanicals
For the orchid grower or trader, one needs to understand the orchids’
cultural requirement in order for the plants to grow healthy at and its best
appearance. These are as follows:
Light. The plant will prefer a slightly brighter location to full sun. Light is
the most important element in the successful production of orchids. Based on the
type of orchid, the plant will prefer exposure to morning sun and could tolerate
direct sun, but must be protected from it during very hot months. Sunlight can be
filtered using 2-3 layers of net 8 feet above the plants during the summer period.
Altitude is also a factor which influence light intensity.
Watering. Orchid plants prefer and tolerates a little bit drier condition. Due
to the plants’ anatomical and physiological structure, the orchid can be watered
every other day or even less, like once every other day (e.g. Vandas and most
monopodial orchids), and once every third day or once a week (sympodial
orchids), just as long as the surroundings and companion plants are kept moist
to provide high humidity. However, as a grower, one has to look at how the
plants reacts to watering, and thus, needs to be adjusted accordingly. If the
plants are becoming dehydrated due to intense heat, it may be much proper to
water them 3-4 times a day. Plants needs to be grouped together based on
watering requirements. Moreover, these plants need to be protected from
excessive monsoon rains through the establishment of a plastic-covered
greenhouse, as prolonged moisture can cause rotting and attract pests and
diseases.
Ventilation. Orchids prefers an area with slight breeze but not a very
windy area, in order to dry some of the moisture in its foliage and potting media
46
during the day. With these, there is little risk that the plant will rot due to excess
moisture. The plant can also tolerate a little bit of dryness as contributed by its
windy surroundings. Ventilation or air movement can be done planning the
position of the greenhouse to the direction of the seasonal monsoon wind or by
providing artificial air movement.
Potting Media & Potting Technique. These plants are best potted on
plastic, clay or hardwood baskets (hanging), tree fern slabs, or in drift woods,
with their root well exposed to air. They can also be grown in coarse brick and
charcoal mixtures in pots on benches, or hanging, in which case they can also be
grown in hardwood baskets with little or no pot-ting mixture required. The roots
are thick and will grow out of the pot or other container; hanging plants often
develop a mass of pendent aerial roots. Such plants do well, but the potting
medium must retain moisture for a particular time, provide nutrients as well as
serve as a stable anchorage for roots..
47
Gubatum Trader – These are group similar to that of the Orchid Trader,
however, they specialize on native orchids and plants. They also have a wide
connection and linkages and are usually located in a particular province. They
collect native orchid plants directly from the forest (usually in areas with logging
activities), together with other epiphytes, ferns, palms, and other ornamental
plants cultivated in the area (e.g. bromeliads, tillandsias, aroids, palms, bonsai,
fruit trees, forest trees, etc.), brings them to Manila or in areas where there are
orchid and garden shows, and sells them on a retail basis. Some have learned
to cultivate and propagate their selected traded plant species and selected
varieties. They are also concentrated on the business aspect of orchids.
Grower / Plant Propagator – These group are those that have sufficient farm
space, those who have a nursery or a green house, have budget and are expert
in mass producing and growing large scale quantities of orchids. They are expert
in growing and flower inducing the plants, and have lots of manpower for the
various farm operations needed for the successful maintenance and fast-
propagation of selected hybrids and species important for trade. Usually they
may either sell on their own or get the help of orchid traders in selling their
produce. They are also concentrated on the business aspect of orchids. For
some, they may specialize in the laboratory (seedlings); in the nursery (seedlings
to mature plants), or in the greenhouse (maintenance and flowering of orchids).
Florist – These are individuals who have artistic inclinations in arranging orchid
cut-flower in bouquets or in arrangements. They may or may not have flower
shops. They usually buy cut-flowers and cut-foliage from growers and offer
flower arrangement services for clients.
Rent-A-Plant Business – These are individuals who may grow their own plants
or just buy plants from growers, repot them on durable and attractive plant
containers, and rents the plants to hotels, offices and business establishments.
The renting of the plants maybe on a weekly, monthly or semi-annual basis.
They are also the ones responsible in maintaining or replacing the plants when
needed.
48
Landscaper – These group buys plants on a whole sale basis and arrange them
in outdoor or indoor landscape gardens. They are usually contractors, and have
linkages with growers / plant propagators, and make arrangement (and plan )
with city developers in the greening or landscaping of both government and
private establishments. They offer their landscaping and maintenance services
on a contract basis.
REFERENCES:
AOS. 2001. Orchid Conservation. (Downloaded from http://www.orchidweb.org)
BARBA, RAMON C. & PATENA, LILIAN F. 1999. R Meidum, A New Medim for Tissue Culture of
Orchids. Philippine Journal of Crop Science 34(S1). Poster Paper presented in the 13th
FCSSP Annual Science Conference, Family Country Homes, General Santos City.
BAUTISTA, N.R. 2000. "Preparing an Orchid Germinating & Reflasking Media for
Embryo Culture." Waling-Waling Review. (Metro Manila: Philippine Orchid
Society, VIII, 2: 6-8)
BAUTISTA, N.R., G.B. TAYLAN, A.B. QUILANG, A.V. CARBONELL & T..S.
BUENAVISTA. 2001. "Abscisic acid-Induced Growth Inhibition of
Dendrobium cv. 'Lunsom Green' (Orchidaceae) In Vitro." Quest. (Vol II,
p.4 ) Mandaluyong City, Philippines: Rizal Technological University.
49
BAUTISTA, N.R.; QUILANG, A.B.; TAYLAN, G.B.; and MADERA, R.F. 2001.
"Anti-Contamination Efficiency of Ethyl Alcohol and Fungicide
In Embryo Culture of Dendrobium cv. 'Anching Lubag X Allan Umaki'
(Orchidaceae)". Quest. (Vol II, p.6 ). Mandaluyong City, Philippines: Rizal Technological
University,
COOTES, JIM. 2001. The Orchids of the Philippines. Singapore: Times Edition
DOST. 2002. Philippine National Science & Technology Plan. Bicutan, Taguig,
Metro Manila: Department of Science & Technology.
KANG, L. C.. 1983. Orchids: Their Cultivation & Hybridization. Rev. ed.
Malaysia: Eastern Universities Press SDN. BHD.
MURASHIGE, T. & SKOOG, F. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol. Plant. 15(3):473-497.
50
PALMER, T. 2001. “Dendrobiums of the Philippines: Traditional Uses.” In:
Orchids of the Philippines (22nd Annual Orchid Workshop, August 4, 2004)
Houston, Texas, USA: The Houston Orchid Society. pp.47
PCARRD. 1994. The Philippines Recommends for Orchids. rev. ed. Los Baños,
Laguna. Philippine Council for Agriculture, Forestry and Natural
Resources and Development Department of Science and Technology
QUILANG, A.B.; BAUTISTA, N.R., TAYLAN, G.B.; MADERA, R.F.; and PULMA,
C.C. 2001. Growth Response of Dendrobium crumenatum (Orchidaceae)
to Knudson C Media with Supplements. Quest. (Vol II, p.11 ).
Mandaluyong City, Philippines: Rizal Technological University
QUILANG, A.B.; BAUTISTA, N.R.; TAYLAN, G.B.; & MADERA, R.F. 2001.
"Growth and Flowering Performance of Dendrobium cv. 'Anching Lubag X
Allan Umaki' (Orchidaceae) to Foliar Fertilizer." Quest. (Vol II, p.5 ).
Mandaluyong City, Philippines: Rizal Technological University.
51
STEWART. JOYCE. Ed. 1992. Orchids at Kew. Singapore: HMSO Publications
Centre.
APPENDIX
APPENDIX A
PREPARATION OF R MEDIA FOR ORCHID
EMBRYO CULTURE
The R Medium was developed by the group of Dr. Ramon C. Barba and Dr. Lilian F.
Patena, Institute of Plant Breeding, University of the Philippines at Los Banos, Laguna,
Philippines.
METHODOLOGY
I. Macroelement Stock 20X Concentration (RM Macro)
2. Dissolve the following chemical reagents one at a time in a glass beaker with 700 ml distilled
water using a magnetic stirrer.
3. When all the salts are dissolve, pour enough distilled water to make a 1000 ml solution.
4. Pour in a brown bottle, and label (Name of solution, Date Prepared). Store stock solution in the
refrigerator.
Note: Use 50 ml of this stock in making 1 L medium.
52
III. Microelements Stock 200 X Concentration (RM Micro)
1. Weigh the following chemical reagents:
a. Manganese Sulfate MnSO4 . H2O 0.13 grams
b. Boric Acid H3BO3 0.020 grams
c. Zinc Sulfate ZnSO4. H2O 0.020 grams
d. Copper Sulfate CuSO4 . 5H2O 0.004 grams
2. Dissolve the following chemical reagents one at a time in a glass beaker with 700 ml distilled
water using a magnetic stirrer.
3. When all the salts are dissolve, pour enough distilled water to make a 1000 ml solution. Pour
in a brown bottle, and label(Name of solution, Date Prepared). Store stock solution in the
refrigerator.
IV. Vitamin & Amino acids Stock 200 X Concentration (RM Vitamins &
amino acids)
1. Weigh the following chemical reagents:
2. Dissolve the following chemical reagents one at a time in a glass beaker with 200 ml distilled
water using a magnetic stirrer.
3. When all the salts are dissolve, pour enough distilled water to make a 250 ml solution.
4. Pour in a brown bottle, and label (Name of solution, Date Prepared). Store stock solution in the
refrigerator.
2. Dissolve the following chemical reagents one at a time in a glass beaker with 200 ml distilled
water using a magnetic stirrer.
3. When all the salts are dissolve, pour enough distilled water to make a 250 ml solution.
4. Pour in a brown bottle, and label (Name of solution, Date Prepared). Store stock solution in the
refrigerator.
Note: Use 5 ml of this stock in making 1 L medium.
53
VI. Ferrous Sesquestrene Stock
1. Weigh 1.25 grams Ferrous Sesquestrene and dissolve in a beaker with 200 ml distilled water
using a magnetic stirrer.
2. When all the salts are dissolve, pour enough distilled water to make a 250 ml solution.
3. Pour in a brown bottle, and label (Name of solution, Date Prepared). Store stock solution in the
refrigerator.
a. RM Macro Stock 50 ml
b. Magnesium sulfate stock 50 ml
c. RM Micro Stock 5 ml
d. RM Vitamins Stock 5 ml
e. Myo-Inositol Stock 5 ml
f. Ferrous Sesquestrene Stock 5 ml
g. Sucrose 20 grams
h. Agar 8 grams
i. Coconut water (Buko type) 100 ml
j. Yeast Extract 1 gram
k. Tomato Puree 10 grams
l. Banana Homogenate (Bungulan) 50 grams
3. In 1 L beaker, place 600 ml distilled water and Agar. Stir and Heat mixture in a stirring hot
plate until it boils. Remove it from the hot plate when the agar has completely melted and
combine it with the rest of the solution.
5. Dispense the mixture in individual culture jars (catsup or mayonnaise bottle). Cap bottles with
plastic or metal caps, then wrap cap with used bond papers and rubberbands.
6. Sterilize in the pressure cooker at 15 psi for 30 minutes. Then place the culture media in the
culture room to cool. Use it after 1 week.
7. Note: R Media is brownish red in color when sterilized with its distinctive odor.
REFERENCES:
BARBA, RAMON C. & PATENA, LILIAN F. 1999. R Meidum, A New Medim for Tissue Culture
of Orchids. Philippine Journal of Crop Science 34(S1). Poster Paper presented in the 13th
FCSSP Annual Science Conference, Family Country Homes, General Santos City.
----------
* If NH4H2PO4 is not available, use (NH4)2HPO4.
54
APPENDIX B
PREPARATION OF VACIN & WENT MEDIA
The original composition of the Vacin & Went Medium is as follows (Vacin &
Went, 1949):
Composition Chemical Symbol Amount (g/L)
Potassium nitrate KNO3 0.525
Ammonium sulfate NH4(NO3)2 . 4H2O 0.500
Tricalcium phosphate Ca3(PO4)2 0.200
Magnesium sulfate MgSO4 . 7H2O 0.250
Potassium phosphate KH2PO4 0.250
monobasic
Ferric Tartrate e2(C4H4O6)3 . 2H2O 0.025
Manganese sulfate MnSO4 . 4H2O 0.0075
Sucrose 20.00
Agar 16.00
NOTE: The Vacin & Went Medium is buffered, and was formulated to solve the problem of
lowering pH after sterilization of the Knudson C medium.
2. In a 1000 ml beaker, dissolve the first chemical (a = ammonium sulfate) in approximately 900
ml distilled water.
3. Bring the volume to 1000 ml in a volumetric flask using distilled water.
4. Place and store the stock solution in a tightly covered brown bottle at room temperature.
5. Repeat the procedure for all the chemicals. Dissolve each chemical separately and place them
on separate brown bottles.
6. Label the bottles with the corresponding chemicals with the name, date prepared and amount
to be used per liter media.
In the preparation of stock solution, Tricalcium phosphate will not usually dissolve. To solve this
problem, make the stock solution more acidic by adding 1 N HCl. The chemical will usually
dissolve at pH = 4.0.
55
There might be problems in dissolving Ferric Tartrate. Instead of the said chemical, Ferrous
sulfate with EDTA can be used instead.
Note: Sodium EDTA is used as chelator for Ferrous sulfate, to prevent it from precipitating.
Ferrous sulfate will not likely to dissolve without sodium EDTA. Use this stock at 10 ml each per
liter of stock solution.
56
APPENDIX C.
MURASHIGE AND SKOOG’S MEDIUM (MS)
The original components of the MS Medium are listed below (Murashige & Skoog, 1962):
This stock solution is 10 times the formula concentration. Use 100 mL of this stock in making 1
liter MS medium.
57
Minor Salts (MS Micro – 100 x)
1. Weigh the following chemical salts:
Milligrams (mg)
Manganese sulfate MnSO4 . 4H2O 1680
Zinc sulfate ZnSO4 . 7H2O 860
Boric acid H3BO3 620
Potassium iodide KI 83
Sodium molybdate Na2MoO4 . 2H2O 25
Copper sulfate CuSO4 . 5H2O 2.5
Cobalt chloride CoCl2 . 6H2O 2.5
2. Dissolve the first 5 chemical salts in 700 mL distilled water, one at at time in a mixing
flask.
3. The two remaining compounds, Copper sulfate and Cobalt chloride, are too small an
amount to weigh accurately on many balances. Thus, weigh 25 mg (a convenient
amount) of copper sulfate and 25 mg of cobalt chloride and dissolve them in 100 ml
distilled water. Ten (10) mL of this solution will contain the desired amount , 2.5 mg each
for the stock solution. Pipet 10 mL of this solution into the mixing flask, together with the
5 chemicals, and save the balance of cobalt / copper solution for further use (store in
refrigerator).
4. Add enough distilled water to make 1000 mL. This final solution is 100 times the formula
concentration.
5. Store in brown bottle. Label and store in the refrigerator.
Note: Sodium EDTA is used as chelator for Ferrous sulfate, to prevent it from precipitating.
Ferrous sulfate will not likely to dissolve without sodium EDTA. Use this stock at 10 ml each per
liter of stock solution.
Sodium ethylene-
diaminetetra-acetate Na2EDTA 0.0373
Ferrous sulfate FeSO4 . 7H20 0.0278
58
VITAMIN STOCK SOLUTION (Optional) (Nitsch & Nitsch, 1969)
Vitamins usually enhances growth of cultures in vitro. Vitamins used in orchid embryo culture
includes Vitamin B complexes which help in the cells' metabolism. Vitamin B complex from drug
stores can also be used.
The vitamin stock solution below is that of Nitsch & Nitsch.
2. Add enough distilled water to make a 500 ml solution in a volumetric flask. Folic acid will not
usually dissolve.
3. Adjust pH to 7.00 to dissolve Folic acid by adding drops of 1 N KOH or NaOH. A clear
transparent solution will result.
4. Store vitamin stock in a brown bottle and inside the refrigerator.
MYO-INOSITOL STOCK
This stock could replace coconut water. To make the stock solution, dissolve 5 grams of
Myo-inositol powder in enough distilled water to make a 500 ml solution. Use 10 ml of this stock
in making 1 liter media. Store the solution in a brown bottle and in a refrigerator.
MS Macro Stock 10 mL
MS Micro Stock 100 mL
Fe-EDTA Stock 10 mL
Vitamins Stock 10 mL
Myo-Inositol Stock 10 mL
Coconut water 100 mL
Sucrose 30 grams
Agar 10 grams
2. Combine the first 7 components in a glass beaker. Mix them briskly to dissolve the
sucrose.
3. Cook and dissolve Agar in 300 mL boiling water. Then add to the rest of the medium.
4. Add enough distilled water to make a 1 Liter medium
5. Adjust pH to 5.6 using 1 N HCl or 1 N KOH.
6. Dispense medium into culture flask, cover and sterilize in pressure cooker.
59
APPEDIX D.
PREPARATION OF PLANT GROWTH REGULATOR
STOCK SOLUTION
Plant Growth Regulators are necessary for tissue culture. They can be prepared by following the
steps below:
Auxin
A. Naphthalene acetic acid (NAA) -- Used for rooting and for multiplication
Concentration = 0.1 mg/L
1. Weigh 25 mg of NAA powder and place in a 500 mL beaker.
2. Using a dropper, add 5 drops of 95% ethyl alcohol, slowly agitating the beaker
until the NAA powder has dissolved.
3. Then, add enough distilled water to make 250 mL stock. This provides a stock
solution which is 100 X Concentration. Each 1 mL contains 0.1 mg of NAA.
B. Indoleacetic acid (IAA) -- Used for rooting, shoot elongation and for multiplication (in
synergistic effect with BA or Kinetin).
Concentration = 0.1 mg/L
1. Weigh 25 mg of IAA powder and place in a 500 mL beaker.
2. Using a dropper, add 5 drops of 95% ethyl alcohol, slowly agitating the beaker
until the IAA powder has dissolved.
3. Then, add enough distilled water to make 250 mL stock. This provides a stock
solution which is 100 X Concentration. Each 1 mL contains 0.1 mg of IAA.
Cytokinin
A. Benzyladenine / Benzylaminopurine Stock (BA or BAP). – used for multiplication
Concentration = 0.1 mg/L
1. Weigh 25 mg of BA powder and place in a 500 mL beaker.
2. Using a dropper, add 1 N Hydrochloric acid (HCl) solution dropwise while stirring
with a glass stirring rod until the BA powder has dissolved.
3. Then, add enough distilled water to make 250 mL stock. This provides a stock
solution which is 100 X Concentration. Each 1 mL contains 0.1 mg of BA.
Giberrelins
A.. Gibberellic Acid Stock (GA3). – Used for shoot elongation or to break
Seed dormancy Concentration = 0.5 mg/L
1. Weigh 50 mg of GA3 powder and place in a 500 mL beaker.
2. Using a dropper, add 1 N Sodium Hydroxide (NaOH) dropwise while stirring win
a glass stirring rod until the GA3 powder has dissolved.
3. Then, add enough distilled water to make 100 mL stock. This provides a stock
solution which is 100 X Concentration. Each 1 mL contains 0.5 mg of GA3.
60
APPEDIX E
PREPARING YAMADA’S MEDIUM
This is a very simple orchid medium made from an orchid fertilizer, which has
made orchid seed sowing simple and attractive to orchid hobbyists.
2. In a glass or plastic breaker, fill it with 500mL water, the drop one chemical
(except the agar) at a time while continuously mixing it with a glass stirrer.
3. In a separate metal kettle or saucepan, mix 400 mL water and the Agar and
sugar. Place the kettle or saucepan in an open flame and stirr occassionally until
the water boils and the agar dissolves. Then pour the mixture into the beaker
with the other chemicals.
4. Add the coconut waer, osterized tomatoes and enough distilled water to make
1000 mL.
5. Test the pH to 5.6 using a pH paper and adjust the pH with 1N HCl or 1N KOH
/ NaOH if it is too acidic or too basic
6. Pour contents into glass jars, then cover with its original plastic / metal cap ,
and then with a piece of paper before sterilizing them in the pressure cooker at
15 psi for 15 - 20 minutes.
61
APPENDIX F
PREPARATION OF WHITE’S MEDIUM
The original components of the MS Medium are listed below
(Murashige & Skoog, 1962):
Adjust PH = 5.5
For every Liter of nutrient medium, add 1 mL of a vitamin stock which contains the following
Glycine 300 mg, Nicotinic acid 50 mg, Thiamine10 mg, Pyridoxine 10 mg per 100 mL of water.
Also add a pH indicator (adjusted to pH 6.0) prepared by dissolving 100 mg chlorophenol red in
25 ml 0.01 N NaOH, then adding water to make 250 mL. The nutrient should have a pH of about
5.5 as indicated by pink color; if it is yellow, it can be adjusted with 0.1 N KOH.
62
APPENDIX G
List of orchids which have been clonally propagated in vitro
63
Lycaste Shoot tip Arditti (1977)
APPENDIX H
64
DIRECTORY OF SUPPLIERS FOR ORCHID GROWING &
LABORATORY
ALYSONS’ CHEMICAL ENTERPRISE, INC.
Supplier of Chemical Reagents and Orchid Fertilizers
1425 G. Araneta Ave., Quezon City
Tel. 712-2266
BELMAN LABORATORIES
Supplier of Chemical Reagents, Orchid Lab Equipments and Orchid
Fertilizers
Belman Bldg. II, 78 Cordillera corner Quezon Avene, Quezon City
Tel. 712-0201 Fax 712-0182
RAMON CALADO
Orchid Seed Culture Services, Supplier of quality orchid seedlings
Sumulong Highway, Antipolo City Tel. 09173364124; 457-8691
65
APPENDIX I
GLOSSARY OF ORCHID TERMS
ACINACIFORM Scimitar-shaped. A "scimitar" is a type of curved sword you see in those 1,001
Arabian Nights type movies. I.E. curved-shaped.
ACRANTHOUS Term applies to sympodial type orchids, referring to the annual portions of
successive growth of the rhizome, each beginning with scaled-leaves, ending with an
inflorescence.
ACROPETAL Leaves and flowers developing successively (one after the other) on one axis so
youngest is at the apex (top).
ACTIVE INGREDIENT any material in a pesticide preparation which is responsible for the killing,
suppression, or control of pests and diseases.
ACULEATE prickle-shaped
ADNATION
adj. Adnate Fusion of unlike parts, e.g. labellum with column; contrasted with connation
ADUMBRAL Shady
ADVENTITIOUS Applied to roots which do not arise from the radicle or its subdivisions, but from
a node on the stem, etc
AGAR "Agar" is just an easier way of saying the real name "agar agar"
AGAR-AGAR Gelantinous substance obtained mostly as translucent strips or white powder from
certain sea weeds; used as solidifying agent in culture media.
ALATA Winged
ALBA Flower with all segments white, but which may have some degree of yellow on the lip only
66
ALLIANCE Designates a group of genera that have many common characteristics and can be
used for cross breeding to produce new hybrid genera. An alliance is limited to genera within a
single tribe.
ALLO Combining form denoting differential characteristics or forms; differentiation from normal
AMABILIS Lovely
AMPHIDIPLOIDS – an organism that is diploid for two genomes, each from different species.
ANASTOMOSE When one vein unites with another, the connection forming a reticulation
ANTHER In seed plants, part of the stamen which develops and contains pollen
ANTHESIS The period between the opening of the flower bud and the withering of the flower or
stamens
ANTICOUS The fore-part, i.e. that most remote or turned away from the axis
ANEUPLOIDY a condition in which all the cells of an organism contain an abnormal number of
chromosome
APICAL At the tip; as in an inflorescence borne at the top of the stem or pseudobulb
APICAL MERISTEM the region of dividing cells at the tip of a plant shoot.
APICULE adj. Apiculate Furnished with a short sharp, but not stiff, point
67
AWARD JUDGING The noncompetitive judging of plants and/or flowers for inherent quality
according to established procedures.
AXIL Upper angle formed between the stem or branch and any other branch, leaf or other organ
arising from them
AXIS 1. Upper angle formed between the stem or branch and any other branch, leaf or other
organ arising from them
2. The main line of growth in a plant or organ, e.g., the stem, from which the other parts such as
the leaves and flowers grow
BACKCROSS To cross or breed a hybrid with one of its parents or with another organism
genetically equivalent to such a parent.
BINOMIAL NOMENCLATURE The system of naming that makes use of two names, the generic
and specific names for each type of organism.
BOTANICAL VARIETY A wild variant warranting botanical recognition and having a status
between subspecies and forma. (Abbreviated as var. or v.)
BRACT A leaf-like organ (often very reduced or absent) bearing a flower, inflorescence or partial
infloescence in its axil
BREEDER The firm or individual who originates a cross and produces progeny for distribution,
irrespective of ownership of parent plants; agent technically concerned in pollination, germination,
etc.
BREEDING The planned production of horticulturally desirable forms through selection, crossing,
and/or hybridization.
BUD An unopened flower; a small swelling or projection on a plant from which a shoot, cluster of
leaves or flowers develop.
BULBOUS Bulb-like
68
BURSICLE A membranous pocket or pouch in the orchid flower, covering or enclosing the
viscidium to stop it from drying up, and being pushed back by visiting insects.
CALLUS pl. calli 1. A waxy or fleshy protuberance on the labellum; 2. A solid protuberance
caused by a mass of cells.
CALPEL The flower part which encloses the ovules and extends into a compound pistil.
CALYX Outside covering, usually green, of flower bud, which splits open as the petals grow
CAPSULE A dry fruit which opens, when the seeds are ripe, at several slits or holes. Any closed
vessel containing spores or seeds.
CARPEL Simple PISTIL, or one member of a compound PISTIL, spore bearing organ.
CAUDATE Having a "tail" or narrowed, apical extension, as some sepals and petals.
CAUDICLE Slender stalk-like appendage that attaches the VISCIDIUM (a sticky gland) to the
POLLINIA (pollen packets).
CHLOROPHYLL The green pigment in the leaves and sometimes stems of most plants, which
uses solar energy to convert carbon dioxide and water to sugar, which is essential in the
manufacture of food by the plants.
CHROMOSOMES The filamentous or rod-shaped bodies in the cell nucleus that bear hereditary
determiners or genes.
CLINANDRIUM A cavity, at the apex of a column in orchids, in which the anthers rest.
CLONE A plant derived by vegetative/asexual propagation from one original specimen, the clone
has identical genotype and phenotypic characteristics as that of its original specimen.
COLUMN The male and female reproductive organs of the orchid. The column (technically called
a "gynostemium") is formed by the fusion of male portion of the flower (stamens) and female
portion (pistils). This is one major characteristic that defines orchids and differentiates them from
all other flowering plants.
COMPOT - acronym for community pots. It is a 4 inches diameter clay pot filled with charcoal
and lined with chopped tree fern or coconut husk on top. It contains about 25-35 orchid seedling.
69
CONNATION
adj. Connate Fusion of like parts. e.g. sepal with sepal: contrasted with adnation.
CREST An elevated and irregular or toothed ridge, in orchids found on the lip
CROSS The progeny resulting from pollination from one plant to another. The term is sometimes
applied to a hybrid between different species. "CROSS" is also used to describe transferring of
pollen from one flower of a plant to another flower of a different plant.
CULTIVAR The horticulture term for "variety" (cultivated variety) used in botany which refers to
minor differences that differentiates a plant from the typical species such as a variation in flower
color.
DECIDUOUS "falling off"; Plants that periodically (usually seasonally) loose their foliage to
conserve moisture during dormant period. E.g. Dendrobium anosmum
DEFLASKING - taking the orchid seedlings our of the flask and to be compotted.
DIFFERENTIATION In a very broad sense, it applies to any situation in shich actively growing
young cells give rise to two or more types of cell, tissue, or organ which are qualitatively different
from each other; the formation of specialized cells and tissues.
DIOECIOUS Unisexual; with the male (staminate) and female (pistillate) flowers on different
individual plants
DIPLOID Having the two sets of chromosomes per cell, designated as 2n.
DIURNAL Referring to daytime; in reference to flowers, signifying those which open only during
the day
DOMINANT GENE A gene is called dominant if its phenotypic effect is the same in the
heterozygous as in the homozygous condition.
70
EMBRYO SAC The female gametophyte of angiosperms. It contains several haploid nuclei
formed by the division of the haploid megaspore nucleus.
EMULSION A dispersion system in which droplets of one liquid are suspended in another liquid,
both liquids being immiscible. Example is oil droplets in water.
ENDOSPERM Triploid nutritive cells surrounding and nourishing the embryo in seed plants.
ENDEMIC SPECIES Species found growing wild in a particular place and particular country and
not found elsewhere.
EPICHIL The upper part of the jointed, complex lip of certain orchids, as in the genus Stanhopea
EPIPHYTE adj. Epiphytic “ipe”, above or on; “phyte”, plant; a plant that grows on another plant--
such as on a bush or tree, but is not nourished by it (hence, not parasitic). They use the host only
for anchorage, drawing food and moisture from the air and from humus collected in the angles of
branches or in the crevices of the bark. An "air-plant." Orchids generally are found growing one of
three ways: as EPIPHYTES (the majority grow in this manner), LITHOPHYTES, or
TERRESTRIALS.
EXPLANT - a piece of tissue, seed or plant part from a mother plant, sterilized and planted in a
flask containing nutrient media for tissue culture.
INOCULATION - process of planting the explant into a flask containing nutrient media.
FAMILY - A group of plants, usually of several genera, and many species, which have the same
basic floral structure and can thus be readily segregated and recognised from other families.
FERTILISATION - The fusion of two gametes to form a new individual (zygote). Cross-fertilisation
refers to male and female gametes from different flowers fusing.
FLASK - A glass container used in the germination of orchid seeds and new seedlings.
FLASKING - This is the process of sowing orchid seeds in a flask or transplanting seedlings into
a flask. It is another word for subculturing; transferring an orchid plantlet or protocorm from an
crowded flask or bottle into a new flask containing fresh new media and the process is intended
for the further growth of the seedling.
71
FOLIACEOUS Leaf-like; used particularly in reference to sepals or bracts which simulate small or
large leaves in texture, size, or colour
FUGACIOUS Withering quickly; falling off soon after anthesis (in reference to a flower)
GENUS
pl. Genera
A classificatory term for a group of plants, usually composed of several slightly different species,
but with characters distinctive enough to enable the genus to be recognised as a separate entity
within a family.
GLANDULAR With glands, secreting organs, often tiny, which usually make the plant sticky.
GLAUCOUS Covered with a bluish-grey, bluish-green, or whitish bloom which will not rub off
GREX A Latin word meaning "group" or "flock"; the name used to describe a group of offspring of
any given hybrid cross. When a grex name is registered, All additional identical crosses, plants
produced from seeds of that cross or any asexual divisions of the cross all have the same grex
name.
"orchid hybrid (grex) names"
The International Orchid Register is the century old international registration system for orchid
hybrids. Its purpose is to ensure that grex nomenclature is uniform, accurate and stable, free from
duplication and in accord with internationallly agreed rules.
The Orchid Review is the first place in which all new grex registrations are published for the first
time, thus providing an important international service to the orchid world.
HAPLOID - an having only half (n) the number of the chromosomes in its cells.
HOMONYM A taxonomic designation rejected because the identical term has been used to
disignate another group of the same rank (a Synonym)
HUMUS The brown or blackish substance, sometimes called vegetable mould, which is the final
result of the decay of organic matter in the soil.
Hybrid - a cross between two species or genera
Inoculation - process of planting the explant into a flask containing nutrient media.
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HYBRID A plant which is the offspring of parents of different species. Hybrids are either
INTRAGENERIC or INTERGENERIC.
The International Orchid Register is the century old international registration system for orchid
hybrids. Its purpose is to ensure that grex nomenclature is uniform, accurate and stable, free from
duplication and in accord with internationallly agreed rules.
The Orchid Review is the first place in which all new grex registrations are published for the first
time, thus providing an important international service to the orchid world.
HYPHAE A threadlike filament possessed by many fungi that function in nutrient absorption and
transfer.
INTERGENERIC Term usually used when referring Cross breeding different species from
different genera producing new hibrids. Genera are always genetically related members of the
same taxonomic Tribe .
INTRAGENERIC Term usually used when referring to cross breeding different species of a single
genera producing new hybrids.
IN VITRO - "in glass"; referring to a plant placed inside a sterile, artificial culture vessel containing
a sterile nutrient media and provided with an artificial / controlled growing environment; growing of
a plant in such a situation.
KEIKI Hawaiian term used by orchidists to signify an offshoot or offset from a plant. Filipino term
is ‘Anak’
LABELLUM - Lip, particularly that for an orchid, a modified lowermost third petal of an orchid
flower, usually where an insect pollinator lands during pollination.
LIP
also labellum A petal, usually of quite different shape and size to the others, normally at the
bottom of the flower, or apparently so, and often, especially in orchids, of complicated structure.
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LITHOPHYTE
adj. Lithophytic litho-, stone; phyte, plant; a plant that grows on stone-- using it for anchorage,
drawing food and moisture from the air and from humus collected in the crevices of the stone. An
"air-plant." Orchids generally are found growing one of three ways: LITHOPHYTIC, EPIPHYTES
(the majority grow in this manner), or TERRESTRIALS.
LYRATE Shaped like a lyre; with an enlarged apical lobe and smaller lower ones
MAQUIS Arid, stony tracts of siliceous soil, covered with shrubs but not trees, such as frequently
found in mediterranean countries.
MEDIUM
Pl. Media – in plant tissue culture, any substance composed of distilled water, mineral salts,
vitamins, sugar, hormones and other organic additives used to grow a piece of plant in vitro; for
orchid cultivation, also known as substrate, the material where the orchid grows or cling into, and
where it gets its nourishment and moisture..
MENTUM The chin-like protuberance occurring in certain orchid flowers, formed usually by the
bases of the lateral sepals with the elongated column-foot
MERISTEM Tissue composed ofDividing cells to produce tissues and organs, located in small
amounts within the growth buds and root tipsThe growing point of shoots.
MERISTEM CULTURE A laboratory technique that involves the taking of the growing meristem
tip from within the new growth and culturing the nucleus of cells, in a similar way to germination of
orchid seeds artificially.
MESOCHILE The intermediate or middle part of the lip of orchids when this structure is separated
into three distinct parts, as in Stanhopea
MONOECIOUS With the male (staminate) flowers and the female (pistillate) flowers borne in
separate inflorescences but on the same plant
MONOPODIUM
pl. monopodia
adj. monopodial Orchids that grow primarily upwards, producing new growth at the top of the
plant from the location of the previous growth. Leaves are produced alternately on either side of
the central stem as it grows. Orchids with a monopodial growth pattern are less common than
those with a sympodial growth pattern.
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MONOTYPIC One type, such as one species in a genus
MOTHER PLANT – a mature orchid plants, usually in bloom, which serves as source of explant,
or seeds, for MICROPROPAGATION .or conventional propagation
MYCELIUM A network of hyphae made from new cells that have elongated and split repeatedly
forming a network. Quiescence- A stage of dormancy that new buds enter during early winter or
periods of cold.
NOCTURNAL Of the night; used in reference to flowers which open after dark
NON-RESUPINATE Orchid flowers normally position the lip at the bottom just above the column.
Some genera, however, such as Cycnoches, Malaxis, and Nephelaphyllum position the lip
uppermost with the column below making the flower appear to be up-side-down.
ORCHIDACEOUS Orchid family, usually having showy flowers with corolla of three petals; one
labellum or lip differs greatly from others and often spurred
PANICLE A branching inflorescence on which all the branches bear flowers, a branched raceme.
PERIANTH Floral envelope, consisting of the calyx and corolla (even if not all parts are present)
the perianth of an orchid flower consists of the sepals, petals, and lip
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PISTIL The female or seed-producing organ of a flower, consisting usually of the ovary, style, and
; in orchid The pistil becomes part of the column and pedicellate ovary
POLLEN Spores or grains borne by the anther, containing the male element; in orchids, it is
usually not granular, as in most other plants
POLLINIUM
pl. Pollinia Coherent masses or "packets" of pollen. Orchids have two, four, six, or eight pollinium
(packets). The number of pollinia is traditionally considered one of the major factors in defining a
genus of an orchid. It is located infront of the column in an orchid flower, usually protected by a
ANTHER CAP.
POLLINARIUM The apperatus of the orchid used to transport pollen from one flower to another.
The pollination consists ofThe POLLINIA (pollen packets), the CAUDICLE (a stalk-lke
appendage), and the VISCIDIUM (a sticky gland)
PROTOCORN The first growth produced by a germinating orchid seed before the growth of
leaves.
PSEUDOBULB Thickened or bulb-like stems (called "pseudobulbs" because they are not true
bulbs) produced by some SYMPODIAL orchids to store water and food. Only orchids whose
habitat has seasonal periods of dryness or drought have adopted this life-saving characteristic.
RACEME A simple unbranched infloresence in which the elongated axis bears flowers on short
stems (pedicels) succession toward the apex.
REVERSE OSMOSIS A process used to purify water by forcing contaminated water through a
semipermeable membrane. The membrane allows the water molecules to pass through but not
the other substances contaminating the water. Reverse Osmosis is used to commercially purify
sea water as well as by hikers to remove impurities from water found along the trail.
RHIZOME The woody parts of the rootstock at the base of the orchid which grows along or just
under the surface of the ground or along host. New growth of sympodial orchids always begins at
the end of the rhizome.
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ROSTELLUM Gr. "little beak": Refers to a slender growth of tissue located at the upper part of the
column which physically seperates the male and female parts thus providing a barrier to prevent
self pollenization. The rostellum also is used to apply a sticky "glue" to the back of the pollinator
(usually an insect such as a bee) to attach the POLLINARIUM (the pollen transport system).
SACCATE With a conspicuous hollow swelling. The term is usually used to describe the bag,
pouch, or sac-shape of the lip on an orchid flower, like the lip-shape of species in genus
Paphiopedilum.
SAPHROPHYTE Plants often lacking chlorophyll; receiving nourishment from dead or decaying
organic matter; needing the services of certain fungi to be able to absorb food.
SCAPE A leafless main flower-stalk arising from the underground or sub-surface parts of a plant
(species of Paphiopedilum are a good example); it may bear scales, bracts, and may be one or
many-flowered
SELFING The pollination by the plant's anthers of either the same flower or a flower on the same
plant. In hybrid names, you will often see (x self) in the name of the plant which means it was
crossed by the same plant. (see SIBBED)
SHEATH The tubular base of the leaf surrounding the flower spike
SIBBED Plants that have the same parentage. In hybrid names, you may see (x sib) in the name.
This means the cross of the plant was made using the same parents. (see SELFING)
SPECIES
pl. species
abbrev. sp. A group of organisms, forming a subdivision of a genus, which have similar
characteristics, enabling one species to be identified from its neighbours; a true species
persistently breeds true to its main characters.
SPIKE An elongated unbranched inflorescence (flower-cluster) in which the flowers are devoid of
pedicels.
STAMEN
pl. stamens
or stamina The male reproductive organ of a flower. In orchids the one or two stamens are part of
the column.
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STELIDIA Column teeth
STEM PROPAGATION Small plants that are formed on flower stems. In some orchids the flower
stem has nodes which carry the dormant eyes and can develop into buds or leaves.These new
plants are called "keiki".There is a hormone compound called keiki paste that is used in the
developmentOf these plantlets. This practice is commonly used on Phalaenopsis.
STIGMA
pl. stigmas
or stigmata The terminal part of the ovary, at the end of the style, which is receptive to the pollen.
STIPE
pl. Stipites A slender, stalk-like stem.
STYLE The narrow portion of the pistil which connects the ovary and the , not usually applicable
to orchids.
SUBGENUS One of the divisions into which large genera are sometimes taxonomically divided.
SYMPODIUM
pl. sympodia
adj. sympodial Orchids that produce new growth from the base of the plant where the previous
growth occured (from the rhizome). The majority of orchids have sympodial growth, the others
have a monopodial growth pattern.
SYNSEPAL A single floral part formed by the partial or complete fusion of two or more of the
orchid sepals (usually the lateral pair as in a Paphiopedilum).
TERRESTRIAL Plants that grow in or near ther surface of the ground; growing in soil. Orchids
generally are found growing one of three ways: as TERRESTRIALS EPIPHYTES (the majority
grow in this manner), or LITHOPHYTES
THROAT In orchids with a tubular lip, used to designate the lower part of the tube
TUBER Thickened and short subterranean branch having numerous buds or eyes.
UMBEL An indeterminate, convex or flat-topped i` in which the pedicels of the cluster arise from a
common point
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UNISEXUAL With flowers of one sex only
URCEOLATE Urn-shaped
VANDACEOUS From the genus name Vanda, alluding to any orchid having the characteristics of
a Vanda species; large monopodial orchids such as genera Aerides, Arachnis Rhynchostylis, and
Renanthera.
VARIETY Plant having minor characters or variations which separates it from the type species.
VEGETATIVE Part of a plant not directly concerned with repoduction as the stem and leaves.
VISCIDIUM
pl. Viscidia A sticky disc-shaped gland located at the base of the caudicle (a slender stalk-like
appendage). Used to attach the pollinia (pollin packets) to an insect allowing the pollen to be
carried to another flower.
WHORL An arrangement of three or more leaves or other organs in a circle about an axis
XEROPHYTE A plant which is adapted to live on a limited supply of moisture, usually in an arid
evironment
ZYGMORPHIC Capable of being divided into two symmetrical halves only by a single longitudinal
plane passing through the axis; all orchid flowers are normally zygomorphic
ZYGOTE Any spore formed by conjunction of two gametes (sex cells); loosely, a zygospore.
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