Documentos de Académico
Documentos de Profesional
Documentos de Cultura
A GUIDEBOOK IN ORCHID
MICROPROPAGATION
Produced by
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
II. Basics in Orchid Anatomy (Lecture)
A. Orchid Growth Habit
B. The Parts & Functions of an Orchid Flower
C. Mechanism of Pollination
D. Embryogenesis
E. Seed Dispersal
F. The Role of the Orchid Mycorrhizae
III. Things needed for the Embryo Culture Technique (Visit the Lab).
A. Chemicals
B. Glasswares and Plastic wares
C. Instruments / tools
D. The Explant
IV. The Technique Proper (Demonstration & Hands-on)
A. Pollination Technique & Pod Harvesting
B. Stock Solution Preparation
C. Media Preparation
D. Sterilization
E. Inoculation
1. Green Pod Culture technique
2. Dry Pod Culture technique
a. Using test tubes
b. Using Petri Dish and filter paper envelope
F. Reflasking
G. Acclimatization
H. Compotting
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
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.
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.
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.
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:
a. embryo culture/embryo rescue - This is the culture of isolated mature or immature
embryoes. These technique is the one primarily used for all orchid types, and the technique was
highly improved by European and American orchid hobbyist in the 1920's in the propagation of
their orchids hybrids. Since orchids have thousands of seeds per capsule, it is the most effective
and efficient way of propagating a endangered or threatened species. The dry pod capsule
technique (using dry matured seeds) where the first technique developed, and then the
revolutionary green pod technique (using immature but fertilized ovules) was developed next.
(Both technique will be discussed in detail in the guidebook). The dry pod technique is used to
produce virus-free seedlings from a virused parent stock. Take note that embryo culture is a
sexual means of reproduction and different from cloning which is asexual.
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
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.
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.
9. Production of Secondary Metabolites - important in medicinal orchids or with scented
flowers or production of pigments; production of pharma-therapeutic important metabolites
(metabolites not used by plant in their growth).
10. Use in Basic Research - for better control of the factors affecting growth: minimize
correlation. This is to improve techniques in micropropagation.
a. Morphogenetic and developmental studies
(1) differentiation of various tissue types (callus cultures); usually affected by
auxins and sugars
(2) organ differentiation - shoots and buds; roots; organogenesis;
(3) Embryogenesis studies - single cells & callus cultures (totipotency & somatic
embryogenesis
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.
10
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.
A convenient location for a small lab is a room or part of of a house, a
garage, a remodeled office or a part of the greenhouse. The minimum area
required for media preparation, transfer and primary growth shelves is about 150
sq ft. Walls may have to be installed to separate different areas.
A good location includes the following:
1. Isolation from foot traffic.
2. No contamination from adjacent rooms.
3.Thermostatically controlled temperature.
4. Water and drains for a sink.
5. Adequate electrical service.
6. Provisions for a fan and intake blower for ventilation.
7. Good lighting.
11
12
13
14
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.
8. Optional equipment -- A variety of non-essential equipment is available for
tissue culture laboratories; individual needs and equipment cost will determine
what can be purchased. Microwave ovens are convenient for defrosting frozen
stocks and heating agar media. Dissecting microscopes are useful to have in the
laboratory for meristeming, dissecting floral and shoot apices, and observing
plant culture growth. Labwashers, or regular dishwashers, can be useful.
Automatic media dispensers are helpful when pipetting large volumes of media.
15
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.
Relative humidity (RH) is difficult to control inside growing vessels, but
fluctuations in the culture room may have a deleterious effect. Cultures can dry
out if the room's RH is less than 50%; humidifiers can be used to correct this
problem. If the RH becomes too high, a dehumidifier is recommended.
Shelving within primary growth rooms can vary depending upon the
situation and the plants grown. Wood is recommended for inexpensive, easy-tobuild 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.
16
17
5. Laboratory Utensils
Pipette Pump / aspirator
Pippetor
Scooper
Knife
Scissors
Scalpel
Forcep
Scraper
Spatula
Microspatula
6. Laboratory Supplies
a. LPG Gas tank
b. Knitted Cotton Gloves
c. Ethyl Alcohol
d. Washing Sponge
e. Soap Detergent
f. Cotton
g. Surgical Blade
h. Waste Basket
i.Fluorescent Lamps
j. Bottle Brush
k. Slippers
l. Mop Head
m. laboratory Gown
n. Hand sprayer
o. Plastic Cover
p. Record Books
q. Aluminum foil
r. Rubber Gloves
s. pH Paper
t. Pitcher
u. Rubbing alcohol
v. Rubber stoppers
w.. Surgical Gauze
x. Fire Extinguisher
y. Rubber Bands
z. Used Paper
aa. Bottle caps
bb.Face mask
cc. Cloth Rugs
dd. Funnel
ee. Marking Pens
ff.Ballast
gg. Pencils
hh. wax paper
7. Media Supplements
Coconut water, tomato puree, banana homogenate (bungulan / saba) , sugar
8. Chemicals (depends on media to be used... for e.g. Knudson C Media)
Chlorox
Lysol
Tween 20
Activated Carbon
Benzyladenine
Kinetin
Naphthalene acetic acid
Peptone
Potassium phosphate
Sucrose
Potassium hydoxide
Copper sulfate
Ammonium sulfate
Ferrous sulfate
Potassium Permanganate
Sodium EDTA
Hydrogen Peroxide
Calcium hypochlorite
Agar
Calcium nitrate
Activated Carbon
Hydrochloric acid
Magnesium sulfate
Manganese sulfate
18
Chapter IV
ASEPSIS: STERILIZATION OF MATERIALS
Sterilizing various plant materials & instruments.
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.
Sterilizing the culture media.
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 :
Time (Minutes)
20 to 50 ml
75 to 225 ml
250 to 500 ml
1000 ml
1500 ml
2000 ml
15 minutes
20 minutes
25 minutes
30 minutes
35 minutes
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.
19
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
OTHER MEANS OF STERILIZATION.
1. Hands --> wash in soap and running water, then with 70% isopropyl / ethyl rubbing alcohol.
2. Glasswares --> can be sterilized in dry oven; cover mouth with aluminum foil sterilize in
pressure cooker
3. Metal Instruments (wire loop, forcep, scalpel etc.--> dry oven; dip in 95% ethyl alcohol, flame in
alcohol lamp
4. Plant Material (Explant) --> surface sterilize with sterilizing solutions. (See below)
5. Glass Petri Dish best sterilized in pressure cooker or hot oven; or another alternative is
inside the laminar flow hood or transfer chamber, pour enough 95% ethyl alcohol (about 10
drops) into an open Petri dish, then flame it by dipping the scalpel or forcep in 95% ethyl alcohol,
flaming it over the alcohol lamp and then placing the flaming instrument into the Petri dish. The
blue flame that ignites the dishs surface is enough to sterilize it. Let it cool first before putting a
plant to dissect since the dish surface is hot.
Concentration used
Calcium hypochlorite
Sodium hypochlorite
Hydrogen peroxide
Bromine water
Silver nitrate
Mercuric chloride
Antibiotics
9-10%
20% v/v
10-12%
1-2%
1%
0.1-1%
4 - 50 mg L
5-30
5-30
5-15
2-10
5-30
2-10
30-60
Very good
Very good
Good
Very good
Good
Satisfactory (toxic)
Fairly good
Concentration Ease of
Used
removal
Treatment
Time (min.)
Sodium hypochlorite
1-1.4%a
+++
5-30
Calcium hypochlorite
9-10%
+++
5-30
Hydrogen peroxide
10-12%
+++++
5-15
Bromine water
1-2%
+++
2-10
Silver nitrate
1%
+
5-30
Mercuric chloride
0.01-1%
+
2-10
Antibiotics
4-50 mg/l
++
30-60
A Common usage rate is 20% v/v of commercial solution
Source: Yeoman & Maclead, 1977.
Remarks
Very effective
Very effective
Effective
Very effective
Effective
Satisfactory
Effective
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.
20
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 crosspollination.
Once pollination is accomplished, it is a standard protocol to remove the labellum or lip of
the flower to discourage other insect pollinator from pollinating the flower. Also, a label is
immediately placed in the flower stalk which takes note the date, and the species or hybrid name
of its parents. For convenience, an accession number or code is used to tag the crosses made,
and all the data of that tag is recorded in a notebook. It has to be noted that a name is very
important for an orchid, specially when you are planning to use it as a parent for your crosses or
when mass producing it. Thus, DO NOT LOOSE THOSE NAMETAGS! For orchid species
collected from the wild or from vendors, it is helpful to get the expertise of your local orchid
taxonomist in identifying your plants.
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.
21
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)
Cattleya
180
Dendrobium
90
Oncidium
70
Aerides
90
Spathoglottis
45
Grammatophylum 90-120
Doritis
Phalaenopsis
Renanthera
Vanda
Paphiopedilum
Cymbidium
90
90-100/120
90-100
120-150
180-240
90-120
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CHAPTER VI
MEDIA PREPARATION AND STERILIZATION FOR
ORCHID EMBRYO CULTURE
STOCK SOLUTION PREPARATION FOR KNUDSON FORMULA C
As of present, there are more than 25 kinds of culture media used for orchids. However, the most
familiar are Knudson's media formula C. Below is the steps in preparing this media. (For other
types of culture media, please refer to the Apendix at the back of the book.)
Preparation of modified Knudson C Stock Solution (20X concentration)
1. Weigh the following chemical salts:
a. Ammonium sulfate
(NH4)2SO4
b. Calcium nitrate
Ca(NO3)2 . H2O
10.00 grams
20.00 grams
5.00 grams
d. Magnesium sulfate
MgSO4 . 7H2O
5.00 grams
e. Manganeses sulfate
MnSO4 . 7H2O
0.15 grams
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.
NOTE: Use 50 ml of each stock solution in preparing 1 Liter culture media.
Preparation of Iron-EDTA Stock Solution (100X Concentration)
1. Weigh the following chemicals:
Ferrous Sulfate Fe2SO4 . 7H2O
Sodium EDTA Na2EDTA
2.78 grams
3.72 grams
23
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.
24
MYO-INOSITOL STOCK
This stock could replace coconut water. To make the stock solution, dissolve 5 grams of Myoinositol 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.
25
50 ml each
20 grams
100 ml
100 grams
8 grams
3 grams
10 ml
10 ml
10 ml
Enough to make 1 L media
Procedure:
1. Get 100 ml of coconut water and place in a 1000 ml beaker.
2. Pour 50 ml each of the Knudson C stock into the beaker.
3. Add 20 grams sugar and mix thoroughly with a glass rod.
4. Add the vitamin stock into the media.
5. Add Myo-Inositol stock into the media
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.
8. Add the activated carbon powder and mix.
9. Add enough water up to the 1 L mark. Mix the media thoroughly.
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.
13. Sterilize the bottles in a pressure cooker at 15 psi for 30 minutes.
14. Place sterilized culture bottles in the culture room to cool.
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CHAPTER VII
MEDIA PREPARATION AND STERILIZATION FOR
ORCHID TISSUE CULTURE
PROCEDURE FOR IN VITRO PROPAGATION OF DENDROBIUM
i.
INITIAL CULTURE
50 mL each
150 mL
10 mL
Enough to make 1 L media
Procedure:
1. Pour 50 mL each of the Knudson C stock into the a 1 Liter beaker.
2. Add 150 mL coconut water (pH should be 5.2 - 5.6)
3. Add 10 mL of 100 ppm BA
4. Add enough water up to the 1 L mark. The solution is mixed thoroughly.
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.
8. Place sterilized culture bottles in the culture room to cool.
Selection, Sterilization and Explant Isolation
1. Select a healthy newly emerging shoot from the base of a Dendrobium
cane.
2. Excise the bud with a sharp knife or scalpel.
3. Wash with detergent and tap water.
4. Swab with 95% ethyl alcohol and rinse with sterile distilled water.
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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.
7. Excise the shot tip and axilary buds.
8. Sterilize explants in 0.5 calcium hypochlorite or 10% bleach for 1 minute.
9. Wash in sterile distilled water.
10. Drop explant in sterile liquid medium.
11. Place culture bottles in rotary shaker
II. PROLIFERATION STAGE
1. Reflask initial culture in liquid medium every 3-4 weeks. Agitate in rotary
shaker.
2. When protocorm-like bodies are formed, transfer a small portion to individual
flasks. Agitate.
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)
4. Protocorm-like bodies may be transferred in a solid medium for more
proliferation with Knudson C + 150 ml coconut water + 1 ppm BA + 7 grams agar.
III. DIFFERENTIATION (Shoot enlargement and in vitro rooting)
Protocorms from liquid medium or the solid proliferating medium may be
transferred to a culture medium with this formulation.
Knudson C + 100 ml coconut water + 50 g. bungulan + 10 g. sugar
+ 7 g. agar (per liter)
Knudson C + 100 ml coconut water + 100 g. bungulan + 20 g. sugar
+ 7 g. agar + 1 g. activated charcoal + 1-5 pm NAA (10-50 ml NAA stock per liter)
28
50 mL each
150 mL
10 mL (for 1 ppm)
10 mL (for 1 ppm)
Enough to make 1 L media
Procedure:
1. Pour 50 mL each of the Knudson C stock into the a 1 Liter beaker.
2. Add 150 mL coconut water (pH should be 5.2 - 5.6)
3. Add 10 mL of 100 ppm BA stock.
4. Add 10 mL of 100 ppm Kinetin stock
5. Add enough water up to the 1 L mark. The solution is mixed thoroughly.
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.
9. Place sterilized culture bottles in the culture room to cool.
29
30
50 mL each
100 mL
20 grams
10 ml (for 1 ppm)
Enough to make 1 L media
Procedure:
1. Pour 50 mL each of the Knudson C stock into the a 1 Liter beaker.
2. Add 100 mL coconut water (pH should be 5.2 - 5.6)
3. Add 20 g. sugar.
4. Add 10 ml of 100 ppm NAA stock.
5. Add enough water up to the 1 L mark. The solution is mixed thoroughly.
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.
9. Incline sterilized test tubes in the culture room to cool.
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32
33
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.
chamber/cabinet, the surface is sterilized by wiping it with 10% chlorox solution.
For transfer
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.
3. The UV lamp and airflow are then turned ON for 30 minutes.
STAY AWAY FROM THE LAMINAR FLOW HOOD WHEN UV LIGHT IS ON, BECAUSE IT CAN
CAUSE SKIN CANCER/EYE DAMAGE!
4. Afterwards, turn off the UV light and bring in the washed dry pods, sterilized Petri dishes,
scalpel, and forcep.
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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.
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.
7. The bottles are then labeled (date and cultivar/species)
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.
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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.
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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.
Some noteworthy orchid species worth collecting:
Amesiella philippinensis, Aerides quinquevulnera, Arachnis longicaulis,
Ascocentrum
miniatum,
Bulbophyllum
spp.,
Dendrobium
anosmum,
Paphiopedilum philippinense, Phaius tankervilleae, Phalaenopsis amabilis,
Phal.equestris, Phal. roeblingiana, Renanthera philippinensis, Ren. monachica,
Spathoglottis plicata, Vanda lamellata, Vanda roeblingiana and a lot more
Some expensive orchids:
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.
Conservation is a very big issue and a word not very much
understood.Wild species (this includes animals, plants and microorganisms) are
protected by CITES (Convention on International Trade of Endangered Species
of Wild Fauna and Flora). This is an international agreement prohibiting or
regulating the trade and sale of threatened or endangered species from one
country into another. It was first organized to protect endangered animals, and
now include plants. Every member country (the Philippines is one of its
signatories) has adopted its own conservation policies patterned after CITES.
The 1992 Conference of the Parties to the Convention on International
Trade in Endangered Species of Wild Fauna and Flora (CITES) adopted
Resolution Conf. 8.19 which called for the production of a standard reference to
the names of Orchidaceae. The orchid genera identified as priorities
(Recommendation 6 prepared by the Royal Botanical Garden, Kew) in the
Review of Significant Trade in Species of Plants included in Appendix II of CITES
(CITES Doc. 8.31) are as follows: Aerangis, Angraecum, Ascocentrum, Bletilla,
Brassavola, Calanthe, Catasetum, Cattleya, Coelogyne, Comparettia,
Cymbidium, Cypripedium, dendrobium, Disa,Dracula, Encyclia, Epidendrum,
Laelia, Lycaste, Masdevallia, Miltonia, Miltoniopsis, Odontoglossum, Oncidium,
Paphiopedilum, Parapahlaenopsis, Phalaenopsis, Phragmipedium, Renanthera,
Rhynchostylis, Rossioglossum, Sophronitis, Vanda and Vandopsis.
The following taxa were listed in Appendix I at the time of publication:
Cattleya trianaei, Dendrobium cruentum, Laelia jongheana, Laelia lobata,
40
elata,
Phragmipedium
spp.,
Renanthera
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.
Here are some conservation recommendations for conserving endangered
and threatened orchid species (including other plants) from the Orchid
Conservation Committee of the Philippine Orchid Society:
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.
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42
leave enough matured plants in the area for future generations. Do salvage
orchids from threatened areas and learn to cultivate them.
Educate native orchid traders on how to take care of the plants.
Traders need to be taught on how to take care of the plants they trade, and to
establish the plants before selling them in the cities. Also, they need to be taught
on how to divide plants for propagation, and also how to establish plants in their
nursery. Also, they need to learn potting techniques, controlling pests and
diseases and how to rejuvenate stressed plants, specially those that are not sold.
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.
Protect Your Collection. In addition to sharing your plants, you should
protect your collections by providing structures or gadgets for the healthy growth
of your plants like a small slat-house or greenhouse, humidifiers, providing
proper ventilation in your garden, a secured fence, practice sanitation and
integrated pest management to prevent disease and pest out-break.
Do not disclose the localities where threatened orchid species are
collected, especially to people whom you do not have utmost confidence. A
sure way to destroy an existing orchid habitat is by reporting nationally or
internationally that a certain orchid species is found in a certain locality. This will
prompt collectors to go to this place over-collect all the orchid plants until there is
nothing left, and sometimes destroying the whole area as well due to colonization
of people.
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.
Educate the people and provide orchid conservation awareness
information through various means. Education is still the best tool to
implement orchid conservation. Conservation advocacy could be done through
print, radio and tv media. The best people to teach are the children and also the
once who are fond of plants.
Create linkages with other conservation groups. People doing
conservation or orchid research could have more advantages by linking or
networking with other groups locally or abroad. They could tap individuals
specializing in certain genera of orchids, institutions doing orchid research,
government or orchid societies. With this, funding, information, and facilities
could be shared.
Be Vocal on Conservation Issues. Be willing to speak out in support of
conservation. This could be in the form of writing letters to editorial boards,
contacting elected representatives, supporting local and national legislation, help
revise government policies which is against conservation and speaking out in
your community on conservation issues. Also, stay active in your local orchid
society, and help it to pursue conservation issues. Report to appropriate
authorities illegal activities that could result in the destruction of orchids and their
habitats, including illegal collection on public lands.
Support Orchid Researches. There is much to learn about orchids. This
includes orchid biology and physiology, taxonomic classification, identifying new
species, information on orchid habitat, orchid geographical distribution,
cultivation, propagation, genetic diversity, pollinators, the orchid-mycorrhizal
relationship, and others. By being part of these researches, information
dissemination or help in allocating funds for such research would greatly help in
orchid conservation.
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.
Main Uses of Orchids
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.
2. Flowering potted plant another use of orchid plants is the production of
flowering potted plants for home and office use. These plants are used as
accents in the home and office, to provide color in center tables, hallways,
corners, lobbies, bedrooms, conference areas, and the garden.
3. Landscape material orchids are now used as a landscaping plant for
public parks, botanical gardens, indoor and outdoor pocket gardens,
especially during orchid and garden shows.
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.
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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..
Fertilization.
In the commercial production of orchids, diluted
commercial orchid foliar fertilizers are highly recommended to be sprayed once a
week, usually after watering in order to provide a regular supply of nutrients to
the plants. Organic fertilizers can also be used, however, make sure that they
are well-decomposed or well-processed or else they may pose problems like
attracting flies, insect pests and diseases. Other forms of fertilizers may include
diluted milk (source of calcium and amino acids), brown sugar (during monsoon
rains), fish emulsions, vermi-cast tea (from earthworms) and others.
Propagation. Orchids can be propagated sexually and asexually. The
best way of propagation is still through production of seeds. Chosen parent
materials are pollinated and allowed to produce seed capsules. Seed capsules
are harvested when mature and are sown in an artificial culture media in the
laboratory till the seeds germinates and develop into complete plantlets.
Asexually, orchids can also be propagated by top cutting, division, or separation
of keikis. . Sterilize all cutting instruments by washing with them in soap and
water and squabbling with isopropyl alcohol before using it to prevent transfer of
viruses. Top-cuts are repotted on plastic or wooden baskets or clay pots with
charcoal. Seal wounds with fungicide paste and divided plants are usually not
watered for 3 days to prevent rotting and for the wound to heal.. Plants can now
be water afterwards to induce establishment of 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 fastpropagation 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).
Hobbyist / Enthusiast These are people with sufficient technical knowledge
and expertise in orchid cultivation, however, they have limited financial
resources. They usually have limited number of plants, maybe 1-10 plants per
kind and are usually grown in the home backyard. They just grow plants for the
sake of the hobby, but some may ocassionally sell their propagations..
Plant Collector These are either Hobbyists / Enthusiasts or Grower, who
collects a specific groups of plants for the joy of the orchid hobby. Some maybe
be able to breed their orchids to produce seedlings. They are often times very
reliable in maintaining plants for very long periods of time.
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)
ARDITTI, J. & ERNST, R. 19__. Micropropagation Of Orchids.
New York: John Wiley & Sons, Inc.
ARQUIZA, AMIHAN M. LUBAG-. 2000. Orchid Micropropagation Training Manual. Ornamental
Crops Division, Department of Horticulture, College of Agriculture, University of the
Philippines at Los Banos, College, Laguna.
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. 1999. "Collecting Orchids for Conservation". Techno Courier.
(Mandaluyong City, Philippines: Research, Extension, & Management Information and
Technology Dissemination Services, Rizal Technological University,
BAUTISTA, N.R. 1999. "Embryo Culture of Orchids." Waling-Waling Review.
(Metro Manila: Philippine Orchid Society, VII, 2: 6-9).
BAUTISTA, N.R. 2000. "Requirements for the Establishment of a Simple Orchid
Embryo Culture Laboratory." Waling-Waling Review. (Metro Manila:
Philippine Orchid Society, VIII, 2: 5-8)
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.
BAUTISTA, N.R. 2001. "Non-Sterile Micropropagation Technique for Orchids ...
Anyone?". Waling-Waling Review. (Metro Manila: Philippine Orchid
Society, IX, 1: 12-16)
BAUTISTA, N.R.; QUILANG, A.B.; TAYLAN, G.B.; MADERA, R.F.; and
PULMA, C.C. 2001. "Effect of Plant Preservative Mixture TM (PPM) on
Contamination Rate and Growth of Vanda sanderiana Reichb.f. Seedlings
(Orchidaceae) In Vitro". Quest. (Vol II, p.9 ). 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,
BAUTISTA, N.R. 2002. "Strategies in the Clonal Mass Propagation of Orchids".
Waling-Waling Review. (Metro Manila: Philippine Orchid Society, X, 2: )
BAUTISTA, N.R. 2002. "The Diverse Orchid Family". Waling-Waling Review.
(Metro Manila: Philippine Orchid Society, X, 1: 12-16 )
BHOJWANI, S. S. & RAZDAN, M.K. 1983. Developments in Crop Science.
Plant Tissue Culture: Theory & Practice. Elsevier Science Publishers
B.V. The Netherlands, U.S. & Canada.
COOTES, JIM. 2001. The Orchids of the Philippines. Singapore: Times Edition
DEL ROSARIO, AURORA G. 1992. Module on Horticulture 113 Plant Tissue
Culture. (unpublished). Department of Horticulture, College of Agriculture.
University of the Philippines at Los Baos, College, Laguna.
DOLERA, NONITO. 2001. Personal Communication. (During one of the meeting
of the Orchid Conservation Network of the Philippines).
DOST. 2002. Philippine National Science & Technology Plan. Bicutan, Taguig,
Metro Manila: Department of Science & Technology.
FESSEL, HANS H. & BALZER, PETER. 1999. A Selection of Native Philippine
Orchids. Singapore: Times Edition & Philippines: VISCA-GTZ Applied
Tropical Ecology Program
GOLAMCO JR., ANDRES S.. 2003. Personal Communication.
HAGSATER, ERIC. 1986. "Can There be a Different View on Orchids
and Conservation?" American Orchid Society Bulletin (March 1986).
pp. 268-271.
KANG, L. C.. 1983. Orchids: Their Cultivation & Hybridization. Rev. ed.
Malaysia: Eastern Universities Press SDN. BHD.
KNUDSON, L. 1946. A new solution for germination of orchid seeds. Amer.
Orchid. Society. Bulletin. 15:214-217.
KOOPOWITZ, HAROLD. 1986. "A Genebank to Conserve Orchids." American
Orchid Society Bulletin (March 1986). pp. 247-250.
KYTE, L. 1987. Plants from Test Tubes. Oregon: Timber Press.
LARSON, R. A. ed. 1980. "Orchids". Introduction to Floriculture.
London: Academic Press, Inc.
MURASHIGE, T. & SKOOG, F. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol. Plant. 15(3):473-497.
ONG, RAYMUNDO G. 2001. Personal Communication.
50
51
APPENDIX
APPENDIX A
METHODOLOGY
I. Macroelement Stock 20X Concentration (RM Macro)
1. Weigh the following chemical reagents:
a. Potassium Nitrate
KNO3
b. Calcium Nitrate
Ca(NO3)2 . 4H2O
c. Ammonium Nitrate
NH4NO3
d. Ammonium Phosphate
NH4H2PO4*
5.0 grams
1.8 grams
3.6 grams
6.0 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.
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.
II. Magnesium Sulfate Stock (Seperated from the Macroelement stock)
1. Weigh 2.0 grams of Magnesium Sulfate (MgSO4 . 7H2O).
2. Dissolve in enough distilled water to make a 1 Liter stock.
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
IV. Vitamin & Amino acids Stock 200 X Concentration (RM Vitamins &
amino acids)
1. Weigh the following chemical reagents:
a. Thiamin HCl
b. Pyridoxin HCl
c. Nicotinic acid
d. Glycine
0.5 grams
0.5 grams
0.25 grams
0.10 grams
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
50 ml
50 ml
5 ml
5 ml
5 ml
5 ml
20 grams
8 grams
100 ml
1 gram
10 grams
50 grams
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
Chemical Symbol
KNO3
NH4(NO3)2 . 4H2O
Ca3(PO4)2
MgSO4 . 7H2O
KH2PO4
e2(C4H4O6)3 . 2H2O
MnSO4 . 4H2O
Amount (g/L)
0.525
0.500
0.200
0.250
0.250
0.025
0.0075
20.00
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.
KNO3
NH4(NO3)2 . 4H2O
Ca3(PO4)2
MgSO4 . 7H2O
KH2PO4
Fe2(C4H4O6)3 . 2H2O
MnSO4 . 4H2O
10.50
10.00
4.00
5.00
5.00
0.50
0.15
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.
NOTE: Use 50 ml of each stock solution in preparing 1 Liter culture media.
55
There might be problems in dissolving Ferric Tartrate. Instead of the said chemical, Ferrous
sulfate with EDTA can be used instead.
Preparation of Iron-EDTA Stock Solution (100X Concentration)
1. Weigh the following chemicals:
Ferrous Sulfate Fe2SO4 . 7H2O
Sodium EDTA Na2EDTA
2.78 grams
3.72 grams
50 mL
50 mL
50 mL
50 mL
50 mL
50 mL
50 mL
20.00
16.00
56
APPENDIX C.
MURASHIGE AND SKOOGS MEDIUM (MS)
The original components of the MS Medium are listed below (Murashige & Skoog, 1962):
Component
Chemical Symbol
Potassium nitrate
Ammonium nitrate
Calcium chloride
Magnesium sulfate
Potassium phosphate
monobasic
Sodium ethylenediaminetetra-acetate
Ferrous sulfate
Manganese sulfate
Zinc sulfate
Boric acid
Potassium iodide
Sodium molybdate
Copper sulfate
Cobalt chloride
Sucrose
Agar
Edamin (optional)
KNO3
NH4NO3
CaCl2 . 2H2O
MgSO4 . H2O
KH2PO4
Na2EDTA
FeSO4 . 7H20
MnSO4 . 4H2O
ZnSO4 . 7H2O
H3BO3
KI
Na2MoO4 . 2H2O
CuSO4 . 5H2O
CoCl2 . 6H2O
Amount (g/L)
1.900
1.650
0.440
0.370
0.170
0.0373
0.0278
0.0223
0.0086
0.0062
0.00083
0.00025
0.000025
0.000025
30.00
10.00
1.00
Chemical Symbol
KNO3
NH4NO3
CaCl2 . 2H2O
MgSO4 . H2O
KH2PO4
Grams
19.0
16.5
4.4
3.7
1.7
57
MnSO4 . 4H2O
ZnSO4 . 7H2O
H3BO3
KI
Na2MoO4 . 2H2O
CuSO4 . 5H2O
CoCl2 . 6H2O
Milligrams (mg)
1680
860
620
83
25
2.5
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.
USE 10 mL of this stock to make 1 L MS medium.
Preparation of Iron-EDTA Stock Solution (100X Concentration)
1.Weigh the following chemicals:
Ferrous Sulfate Fe2SO4 . 7H2O
Sodium EDTA Na2EDTA
2.78 grams
3.72 grams
Na2EDTA
FeSO4 . 7H20
0.0373
0.0278
58
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.
10 mL
100 mL
10 mL
10 mL
10 mL
100 mL
30 grams
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.
C. Indole-3-Butyric Acid (IBA). Used for multiplication (optional).
Concentration = 0.1 mg/L
1. Weigh 25 mg of IBA powder and place in a 500 mL beaker.
2. Using a dropper, add 1 N Sodium Hydroxide (NaOH) solution dropwise while
stirring win a glass stirring rod until the IBA 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 IBA.
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 YAMADAS 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.
Modified Yamadas Medium
Gaviota 67 Fertilizer
Peptone
Agar
Sugar
Coconut water
Fresh tomato extract
Distilled water
1.5 g
1.75 g
10 g
15 g
200 mL
10 mL
1000 ml
PROCEDURE:
61
APPENDIX F
PREPARATION OF WHITES MEDIUM
The original components of the MS Medium are listed below
(Murashige & Skoog, 1962):
Component
Chemical Symbol
Amount (g/L)
Potassium nitrate
Calcium nitrate
Magnesium sulfate
Sodium phosphate
Monobasic
Potassium chloride
Sodium sulfate
Ferric sulfate
Manganese sulfate
Zinc sulfate
Boric acid
Potassium iodide
Molybdic acid
Copper sulfate
Sucrose
Agar
KNO3
Ca(NO3)2 . 4H2O
MgSO4 . H2O
0.080
0.300
0.720
KCl
Na2SO4
Fe2(SO4)3
MnSO4 . 4H2O
ZnSO4 . 7H2O
H3BO3
KI
MoO3
CuSO4 . 5H2O
0.0165
0.065
0.200
0.0025
0.007
0.003
0.0015
0.00075
0.0000001
0.000001
20.00
5.0
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
Explant
Anacamptis pyramidalis
Shoot tip
Morel (1970)
Aranda
Goh (1973)
(Arachnis hookeriana
x Vanda lamellate
Shoot tip
Aranthera
Shoot tip
Arundina bambusifolia
Mitra (1971)
Ascofinetia
Inflorescence segment
(with flower primordial)
Brassocattleya
Axillary bud
Kako (1973)
Calanthe
Shoot tip
Bertsch (1967)
Cattleya
Shoot tip
Axillary bud
Lateral bud
Leaf base
Leaf tip
Cymbidium
Shoot tip
Dendrobium
Shoot tip
Nodal Segment
Flower stalk segment
(with vegetative buds)
Reference
Epidendrum
Leaf tip
Laelia
Axillary Bud
Arditti (1977)
Laeliocattleya
Axillary Bud
Arditti (1977)
63
Lycaste
Shoot tip
Arditti (1977)
Miltonia
Shoot tip
Arditti (1977)
Neostylis
Inflorescence segment
(with flower primordial)
Arditti (1977)
Neottia nidus-avis
Root
Champagnat (1971)
Odontioda
Shoot tip
Arditti (1977)
Odontoglossum
Shoot tip
Arditti (1977)
Odontonia
Shoot tip
Arditti (1977)
Oncidium
Shoot tip
Bertsch (1967)
Oncidium papilio
Fast (1973)
Phajus
Shoot tip
Arditti (1977
Phalaenopsis
Rotor (1949)
Intuong et al. (1972)
Pieper and Zimmer (1976)
Intuong and Sagawa(1974)
Pieper and Zimmer (1976)
Shoot tip
Leaf segment, stem
segment and root segment
Pieione
Shoot tip
Rhynchostylis gigantean
Schomburgkia superbiens
Lateral bud
Arditti (1977)
Vanda (Strap-Leaf)
Shoot tip
Stem section
Vanda Hybrid
(V.teres x V. hookeriana)
Shoot tip
axillary bud, root segment
Goh (1970)
Vascostylis
Inflorescence segment
(with flower primordial)
Arditti (1977)
Vuylstekeara
Shoot tip
Arditti (1977)
APPENDIX H
64
65
APPENDIX I
GLOSSARY OF ORCHID TERMS
ACAULESCENT Having no visible stem, or a very short one
ACAULIS Having no stem
ACCRETE Grown together
ACICULAR Needlelike spine; pointed; bristle
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
ACUMEN A tapering point
ACUTE Distinctly and sharply pointed, but not drawn out
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
AERIAL ROOTS Borne above potting surface
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
ALBESCENT Becoming white or yellow
ALBINISM Lack of color; deficient in pigment
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.
AMPLEXICAUL Clasping the stem
ANAEROBIC Living in the absence of free oxygen
ANASTOMOSE When one vein unites with another, the connection forming a reticulation
ANGULATE More or less angular
ANTHER In seed plants, part of the stamen which develops and contains pollen
ANTHER CAP A dome-shaped structure, protecting the pollinia in an orchid flower.
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
ANTRORSE Turned backwards, directed upwards
ANEUPLOIDY a condition in which all the cells of an organism contain an abnormal number of
chromosome
APHYLLOUS Without leaves
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
APICULE A short and sharp, but not stiff, point
APPLANATE Flattened out or horizontally expanded
APPRESSED Lying flat for the whole length of the organ
ARCUATE Curved like a bow
ARISTATE Tipped with bristle-like appendage or awn
AURICLE A small lobe or ear
AUTOGAMOUS Self-fertilized; flowers that are fertilized by their own pollen.
AUTOPOLYPLOID a polyploid that originates by the multiplication of one basic set of
chromosomes.
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.
BARBATE Bearded; barbed
BASAL At the base of an organ or part such as the stem or pseudobulb
BIFOLIATE With two leaves
BIGENERIC Applies to hybrids made between members of two genera
BINOMIAL NOMENCLATURE The system of naming that makes use of two names, the generic
and specific names for each type of organism.
BISEXUAL Two-sexed; with both stamens and pistils
BLADE Expanded portion of a leaf or petal
BLOOM An individual flower.
BOTANICAL A species or hybrid which, though lacking in horticultural importance, has an
educational value, and the display of which contributes to the dissemination of knowledge of
orchids; any species of orchids which is not grown commercially for its flowers.
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
BRACTEATE Bearing bracts
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.
CADUCOUS Falling off at early stages, when buds fall off.
CAESPITOSE Growing in tufts or dense clumps
CALCAREOUS Containing calcium carbornate, or calcite, chalky.
CALCEOLATE Slipper-like; with the form of a round-toed shoe
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).
CELL The basic unit of life, both in structure and in function.
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.
CILIATE fringed with usually small hairs
CLEISTOGAMOUS With fertilization taking place within the unopened flower
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.
COLUMN-FOOT A basal extension of the column to which the labellum is attached.
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.
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CONNATION
adj. Connate Fusion of like parts. e.g. sepal with sepal: contrasted with adnation.
COROLLA Inner of two series of floral leaves; petals
COTYLEDON Seed-leaf; primary leaf or leaves in an embryo
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.
CUTTINGS section of a plant, usually a stem, used in propagation.
CYTOPLASMIC INHERITANCE Non-Mendelian inheritance involving replicating, cytoplasmic
organelles like viruses, mitochondria, plasmids, etc.
DAMPING OFF The collapse of seedlings, usually caused by infestations of a fungi
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.
DEHISCENCE Spontaneous opening of a ripe fruit to discharge its seeds
DIANDROUS With two stamens, as members of the orchid sub-tribe Cypripedilianea
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.
DISTICHOUS In two ranks or rows on opposite sides of an axis
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.
ECALCARATE Without a spur or spurs
ECHINATE Furnished with prickles or bristles
EMBRYO An organism in early stages of development.
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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 Confined to certain regions, such as country or island.
ENDEMIC SPECIES Species found growing wild in a particular place and particular country and
not found elsewhere.
EPHEMERAL Lasting only one day when in flower
EPICHIL The upper part of the jointed, complex lip of certain orchids, as in the genus Stanhopea
EPICHILE Terminal lobe of labellum in certain orchids
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.
EPITHET The part of a taxonomic name designated a species
EROSE With the margin irregularly notched, as if gnawed
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.
HYBRID - a cross between two species or genera
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.
FASCICULATE - Bundled; radiating from a central growing point.
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.
FLORA - The vegetation or plant life of a given region
FLORIFEROUS - Free-flowering; easily brought into flower
FOETID - With a disagreeable odour
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FOLIACEOUS Leaf-like; used particularly in reference to sepals or bracts which simulate small or
large leaves in texture, size, or colour
FRINGED Furnished with hair-like appendages on the margins
FUGACIOUS Withering quickly; falling off soon after anthesis (in reference to a flower)
FUSIFORM Spindle-shaped, tapering at each end, cigar-shaped.
GENERIC Of or pertaining to a genus
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.
GLABROUS Without hair or down.
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
GREGARIOUS Growing together in clusters or colonies
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.
GYNOSTEMIUM The technical term for the orchid's column.
HAPLOID - an having only half (n) the number of the chromosomes in its cells.
HASTATE Spear shaped, with the basal lobe turned outward
HERBACEOUS Herb-like; not woody
HERBARIUM A collection of dried (or otherwise preserved) plant specimens
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.
HYBRIDIZATION To produce hybrid offspring by pollination; to interbreed; to cross
HYPHAE A threadlike filament possessed by many fungi that function in nutrient absorption and
transfer.
HYPOCHILE Lower or basal part of the lip in some orchids, as in Stanhopea
INDEHISCENT Not splitting open at maturity; opposite of dehiscent
INDIGENOUS Native; not introduced; not exotic
INFLORESCENCE The "flower-cluster";Technically, it's "a general arrangement and disposition
of the flowers on an axis" There are many types of inflorescences based on the form of the flower
cluster and the manner/sequence of flower blooming. The major orchid inflorescence forms
include Spike, Raceme and Scape. Other less common forms seen in orchids include Cyme,
Corymb, and Umbel.
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 .
INTERNODE Potion of a stem situated between the nodes or joints
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.
ISTHMUS A narrowed portion of a part or segment of a flower
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.
LABIATE Lipped; furnished with a lip
LINEAR Narrow and comparatively long, with parallel margins
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.
LOBE A part of a segment that represents a division to about the middle
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.
MARL A chalky clay soil.
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..
MEMBRANACEOUS Thin and more or less translucent
MENTUM The chin-like protuberance occurring in certain orchid flowers, formed usually by the
bases of the lateral sepals with the elongated column-foot
MERICLONE An exact genetic copy of another plant produced by meristem culture.
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
MICROPROPAGATION - A term referring to plant tissue culture technique, an artificial and
aseptic way of plant propagating.
MONANDROUS With one stamen
MONOCOTYLEDON With a single cotyledon or seed-leaf
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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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)
PRICKLE Small sharp spine or thorn
PROGENY Plants grown from seeds produced by parent plants; offsprings
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.
PUBESCENT Hairy, the hairs short, soft and downy
QUADRIGENERIC Pertaining to four genera; used particularly in reference to hybrids combining
members of four genera
RACEME A simple unbranched infloresence in which the elongated axis bears flowers on short
stems (pedicels) succession toward the apex.
RACHIS The axis of a spike, raceme, or branch of a panicle.
RADICAL Belonging or pertaining to the root or base
RAMIFICATION The mode or style of branching of a plant
REPENT Creeping, and typically rooting at the joints
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.
RINGENT Gaping; said of lipped flowers with an open throat or mouth
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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).
ROSETTE A more-or-less dense basal cluster of leaves
RUPICOLOUS Dwelling in or on rocks or stones
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.
SAXICOLOUS Dwelling in or near rocky places; growing on rocks
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
SECUND To one side, as flowers on an inflorescence.
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)
SEPAL The outermost whorl of flower parts.
SESSILE Without a stalk
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.
SPERMATOPHYTE A seed-producing plant
SPIKE An elongated unbranched inflorescence (flower-cluster) in which the flowers are devoid of
pedicels.
SPUR Hollow sac-like or tubular extension of the lip, usually nectariferous
spurred -having spurs
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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