A Guidebook in Orchid Micropropagation

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THE PLANT BIOTECHNOLOGY PROJECT "Conservation of Philippine Native Orchids"  Research & Development Center  Rizal Technological University Boni Avenue, Mandaluyong City 1550 Philippines Telephone: (632))534-8267 Local 135 Email: [email protected] Email: [email protected] ISBN ________________  THE ORCHID MICROPROPAGATION GUIDEBOOK COMMITTEE: TERESA S. BUENAVISTA BUENAVISTA - R&D Director/Project Mgr.  ALEXANDER B. QUILANG - Project Leader  NORBERTO R. BAUTISTA - Co-Project Leader  RACHEL F. MADERA MADERA - Project Staff  CARNETTE C. PULMA - Project Staff  JOVITA A. ANIT ANIT - Project Staff  Staff 

(c) 2010 4th Revised Edition / E-Book Version Edited by Norberto R. Bautista Rizal Technological University, Boni Avenue, Mandaluyong City, 1550 Philippines

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OUTLINE OF TOPICS I. Introduction on Orchid Growing (Lecture).  A. The value of orchid species and hybrids B. Brief History of Orchid Embryo Culture C. An introduction to Plant Tissue Culture 1. The Laboratory 2. The Culture Media 3. The Explant 4. The Techniques 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

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Chapter I INTRODUCTION TO ORCHIDS Orchids are one of the most diverse and advance groups of plants on planet Earth. Its size ranges from the tiny and miniscule botanicals to the gigantic forest epiphytes; with flowers of  various shapes, sizes color and scents, and inhabiting a wide degree of environment. Orchid habitats are widely distributed and they are found in almost all continents except Antarctica.

What’s in An Orchid Name? The name "orchid" came from the first named orchid plant, Orchis  (most likely from Orchis morio  L.). The name means "testicles" in Greek, which makes it more masculine. The name refers to the paired underground bulbs of the Mediterranean orchid because of its similarity to the male reproductive organ. Also, it is practiced in olden times that plants resembling a particular human organ or part, is believed to be a cure for ailments of that part. With this name, orchids are definitely not feminine. The name was used by the Greek philosopher Theophrastus from Lesbos (372-289 BC), in his Enquiry into Plants. Dioscorides adopted the word "orchis" to name two (2) plants which probably referred to the same "orchis" of Theophrastus, in a manuscript of medicinal plants. He also expounded on the aphrodisiac properties of orchids, a concept which lasted until the 15th to 16th century. Thus, the first named orchid was believed to be an aphrodisiac and could revitalize reproductive fertility. Finally, John Lindley used the term to name the orchid family in his book "The Genera and Species of Orchidaceous Plants" (18301840).

Traditional Uses of Orchids  At present, orchids are used as cut-flowers (a multi-million dollar business), house plants and for landscaping. However, there are other uses of orchids aside from these. The orchid Vanilla planifolia, which is native to Central America, and is cultivated in the West Indies and Java for its vanillin flavor. The unripe capsule (fruit) is collected from the plant and dried. The vanillin crystallizes on the outside of the pods. In the past, orchids have been used for a wide variety of  both spiritual and material purpose. The Philippine local folks have an extensive use for orchids, specially the genus Dendrobium (Palmer, 2001). Dendrobium taurinum was used to make a wash to remedy loss of  hair. A tonic decoction was made from Dendrobium crumenatum and it was also used for ear  ache and ear infections. The old stalks from this orchid were cut and used as ties. The dried stems of Dendrobium heterocarpum were used to make a belt to hold up the loincloth. The stems or canes of several species of Dendrobium, including D. macrophyllum, have had local use in various aspects of weaving, basketry and wickerwork. D. crumenatum is used is used for straw plaiting and making straw hats. The yellow material used to decorate artifacts is provided by local species of Dendrobium. This is due to the fact that the orchid stems turn yellow on drying, the color being intensified by exposing the stems to heat from the sun or fire. The outer covering of  the stems are cut into strips, which are either woven into artifacts such as mats and armbands, or  fixed around objects such as arrows and fire sticks. The many types of articles decorated in this way include domestic implements, clothing, body ornaments, cremonial articles, funerary relics and weapons. The yellow strips made from D. crumenatum stem were used for decorative purposes in basketry and hat making, and D. heterocarpum  was used to decorate items of clothing. D.

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tetraedre  is used in small hand-woven baskets and in cigar cases. Yellow bark from D. secundum is used to decorate bows and arrows, personal ornaments and funerary relics.

Modern Uses of Orchids Orchids are primarily grown because of their beautiful flowers. They are mass produced by the thousands or millions to support the world cut-flower industry. Also, interest in orchids arose due to their exotic beauty, rarity, and hidden mystery. Orchids are also raised as rare house plants, wherein most of their owners are orchid specialists and hobbyists. In their native habitat, orchids are used by the country's town folks in many ways. Flowers are used in wedding ceremonies, in honoring guests, or as offering in burial; the seed capsule of Vanilla is used for  flavoring; some of the orchid petals are used as garnishing in dishes; and some orchids with scented flowers are even used as an aphrodisiac, sexual stimulant or as herbal medicine. Orchids are also produced in large numbers as a landscape plant, potted house plant used to accent particular areas in the home. Small flowered orchids nowadays are now gold or silver  electroplated to be made into fine life-size jewelry for women.

The Orchid Hobby  Orchid growing is the most oldest and most stable plant hobby in the world. No plant has so many breeders both amateur and professional. The hobby, often referred to as the "orchid bug" is highly contagious. Orchid enthusiasts usually "infect" neophytes to pursue hobbies in orchid growing or to do serious orchid business. As more and more people are becoming interested in growing orchid and in orchid business, orchid societies which cater to the needs of  these group of people are also created. The orchid industry is also supporting a lot of industries like the clay and plastic pot manufacturers, native orchid collectors, plant propagators, orchid hanger maker, fertilizer and pesticide manufacturers & distributors, driftwood suppliers, charcoal manufacturers and others. Various orchid clubs has also been created due to the hobby. Serious Filipino orchid collectors bring into the country thousands of dollars worth of orchids, building large nurseries and greenhouses just to house their valuable collections.

World Orchid Distribution Orchids are practically found in almost all continents of the world except Antarctica. Orchids has colonized the Earth for thousands of years, and are found from cloud forests to lowlands, tundra to desert. However, a large number of orchid species are found in tropical forests near the equator. Orchids have even adapted in ways that amazed even the naturalist Charles Darwin. An astonishing 35,000 orchid species distributed in 800 genera, have evolved, with remarkable structures and diverse mechanisms for drought resistance, nutrient conservation, pollination and reproduction that even today are not fully understood. The number of species constitutes roughly 10% of all flowering plants in the plant kingdom. In addition to the species are many hybrids and cultivated forms which continue to increase at a steady rate year after year. The Philippines is home of a diverse orchid flora which is composed of more than a thousand species. However, only a few of them are cultivated as ornamentals or for cutflowers, and some have become ancestors of the colorful orchid hybrids of today. Some of the species are valued as botanicals and have inconspicuous short lived flowers. Some of the popular orchid genera grown by hobbyists are  Aerides, Arachnis, Ascocenda, Cattleya, Cymbidium, Dendrobium, Doritis, Grammatophylum, Oncidium, Phalaenopsis, Paphiopedilum, Renanthera, Spathoglottis, Trichoglottis, and  Vanda.  Some well know inter-generic hybrids are

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Brassolaeliacattleya, Kagawara and Mokara. With the ideal conducive climate the Philippines has, both tropical and semi-temperate orchids could be grown in the country, making the archipelago an ideal propagating area for orchids.

Orchid Propagation In the early years of orchid culture, people have no idea of how to propagate these plants. Orchid hobbyist usually get their orchids from forests. Then, they found out that orchids also produce fruits (capsules) when their flowers are pollinated naturally by bees or artificially by man. Each orchid fruit contain thousands of seeds. Orchid seeds are very small (about 470-560 microns long, 80-129 microns wide) and weight about 6 micrograms. Their size is one of the factors which gave them the tendency to be disperse by wind for thousands of kilometers, away from their original habitat. However, not all of  these seeds germinate and grow into mature plants in nature. Most (but not all) orchid seeds need a symbiotic fungus or mycorrhizae, usually of the genus Rhizoctonia, in order to germinate. The fungus needs to infect the seeds in order for it to survive in its early stages of development. The mycorrhizae infects the basal part of the seed and release an enzyme which converts starch in the surrounding area into simple sugars. These will be the energy source or food of the germinating embryo up to its development into a mature plant. Not all of the seeds grow in nature. Only the seeds which land on a suitable surface (a rock, a bark of a branch or on the ground) and infected with its particular mycorrhizae, germinates and grows into a plant. This comprise only about an average of only 1%. However, through science and technology, almost 99% of the seeds could now be raised into mature plants through embryo culture. Embryo culture or embryo rescue is one of the techniques used in the commercial breeding and propagation of orchids. Thousands of plants could be produced in this method in a year, due to the fact that orchids literally produce thousands to millions of seeds (about 6,200 in Cephalanthera grandiflora and 2-3 million in Cattleya labiata). In this method the viable seeds or  ovules are sterilized and placed inside a flask containing artificial growing media. The media consist of mineral salts, vitamins, amino acids, sugars and growth hormones. After a year or so inside a flask, seedlings are then brought out into the nursery where they grow into maturity. From these plants that are produced, a breeder usually selects the best flowering plant, registers it and then clones this selected plant through the conventional division, cutting or kiekis method, or better still, through plant tissue culture. Plant tissue culture, particularly meristem culture or mericloning is the most efficient way of mass producing a selected species or hybrid. In this method, the shoot tip or very young inflorescence is severed, sterilized, and its actively growing region is obtained and cut into minute pieces and inoculated into a flask containing artificial culture media. The tissue is permitted to undergo callus formation, and then grows into minute orchid plantlets. These callus and plantlets are then further divided to produce the required number of plants inside the laboratory. When the right number of plants are obtained, then, the plantlets are then hardened and transferred into the nursery where they are permitted to grow into maturity. The plants produced in these way are true-to-type and identical to that of the mother plant.

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Chapter II INTRODUCTION TO PLANT TISSUE CULTURE Plant tissue culture is one of the biotechnological tool used in the mass propagation of  high value crops, specially orchids. Plant tissue culture is a broad term, which means the growing or cultivation of plantlets or plant parts in an artificial culture medium under aseptic conditions. It is a generic name which includes the following: 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

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also be used in cell fusion, creation of genetically modified orchids (e.g. bombardment with gold particles coated with foreign DNA), or production of secondary metabolites (like scents or  pigments). h. protoplast culture - This involves the culture of naked cells (plants cells devoid of their cell walls); which is a prerequisite for cell fusion, DNA insertion or for counting chromosomes (karyotyping). The cells usually comes from a cell suspension culture, and could also regenerated back into complete whole plants.

APPLICATION OF PLANT TISSUE CULTURE FOR ORCHIDS. 1. Micropropagation   - a rapid technique in multiplication of orchids; more on mass clonal reproduction of orchids with outstanding characteristics. 2. Disease Elimination  - recover plants from pathogen (usually of viral diseases, but also to recover from bacterial and fungal diseases); thus, what is produced are disease free plantlets. Primary technique used is meristem culture. 3. Embryo Rescue  - artificial culture of immature seeds (ovules); as long as pollen tube has fertilized egg. There are many advantages: a. For shorter breeding cycle --> e.g. dendrobium orchids 4-6 months for capsule to mature, but 3 month old (about 75% of the waiting time) capsules can be used for ovule culture. b. To prevent abortion of interspecific or intergeneric crosses 4. Germplasm Conservation & Exchange a. Germplasm collection - for vegetative propagated (sterile or with no seeds) to minimize culture space; and ease in transportation/distribution b. Germplasm conservation - in vitro gene banks; slow growth techniques through use of  inhibitors like growth retardands like Abscisic acid, cold temperature, other retardants like mannitol and sorbitol; cryopreservation (freezing of cell in liquid nitrogen - to achieve suspended animation). c. Germplasm exchange - no more quarantine restrictions since in vitro plants are sterile; also less bulky. 5. Production of Polyploid Plants   - through the use of colchicine or other chemicals which could induce chromosome doubling; polyploid orchids are produced. Colchicine is usually incorporated into the culture media where the seeds will be grown. Polyploid orchids are known to have a much thicker petal and larger flowers. 6. Induced Somaclonal Variation - it usually enhance variability of plants regenerated in tissue culture; usually mutation (polyploidy, deletion, inversion, crossing over) occurs in response to genetic instability while in vitro, or exposure to gamma radiation or to mutagenic substance (e.g. high levels of auxins and cytokinins). Mutation occurs since cells in vitro undergo rapid cell division ... then exposed to mutagens. This is usually a disadvantageous in cloning; but advantageous for crop improvement. Another is the selection of plant variants which has specific characteristics like new flower color, stunting or gigantism; pest tolerance; acid soil tolerance, drought tolerance (different levels of polyethylene glycol) cold tolerance, heat tolerance, herbicide tolerance, and disease resistance. The plant is usually subjected to stress, the ones which survive is said to have the tolerance to the specific stress. 7. Somatic Hybridization - allows asexual additions of heterozygous genomes without meiotic recombination. In protoplast fusion, enzymes are used to devoid cell walls from cells; then allowed to fuse with the use of mannitol. Naked cells readily fuse irrespective of species.

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8. Genetic Engineering  - this involves the formation of new combination of genetic material. Done through insertion of foreign DNA into target cell, use of genetic bombardment gun with gold particles coated with DNA; Other vectors include Agrobacterium, DNA or RNA virus, T plasmid or  pure DNA (direct gene transfer through DNA uptake, microinsertion or electroporation. Then target cell permitted to develop into whole organism which will express the gene. Gene usually with anti-biotic resistance markers. 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.

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CHAPTER III DESIGN FOR AN ORCHID LABORATORY Success in orchid plant tissue culture depends upon various factors like available facilities, choice of nutrients, type of growth regulators, type of explant used, time of explant collection, intensity, duration and type of light, temperature and other variants. The most important factor is that all operations require aseptic conditions, thus absolute cleanliness and orderliness must be maintained throughout the laboratory. Sterilization techniques should be meticulously followed to avoid any contamination of the samples. Design of the tissue culture laboratory usually is specific and fitted with filtered air inlets and decontamination facilities as well as temperature and humidity controls. Because of the rapid advances in this field, there is an increasing need for  training and developing skills in the techniques required for modern plant biotechnology and its applications. In designing any laboratory, big or small, certain elements are essential for  a successful operation. The correct design of a laboratory will not only help maintain asepsis, but it will also achieve a high standard of work.

FACILITIES

Careful planning is an important first step when considering the size and location of a laboratory. It is recommended that visits be make to several other  facilities to view their arrangement and operation. A small lab should be set up first until the proper techniques and markets are developed.  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.

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Larger labs are frequently built as free-standing buildings. Although more expensive to build, the added isolation form adjacent activities will keep the laboratory cleaner. Prefabricated buildings make convenient low cost laboratories. They are readily available in many sizes in most parts of the country. Built-in-place frame buildings can also be used. Consideration should be given to the following: 1. Check with local authorities about zoning and building permits. 2. Locate the building away from sources of contamination such as a gravel driveway or parking lot, soil mixing area, shipping dock, pesticide storage, or dust and chemicals from fields. 3. A clear span building allows for a flexible arrangement of walls. 4. The floor should be concrete or capable of carrying 50 pounds per square foot. Walls and ceiling should be insulated to at least R-15 and be covered inside with a water-resistant material. 5. Windows, if desired, may be placed wherever convenient in the media preparation and glassware washing rooms. 6. The heating system should be capable of maintaining a room temperature at 70 F in the coldest part of winter. 7. A minimum 3/4 in. water service is needed. 8. Connection to a septic system or sanitary sewer should be provided. 9. Air conditioning for summer cooling may be necessary. 10. Electric service capacity for equipment, lights and future expansion should be calculated. A minimum 100 amp service is recommended.

GENERAL LABORATORY DESIGN

Cleanliness is the major consideration when designing a plant tissue culture laboratory. Most companies are not aware of their losses from contamination, but estimates run from less than 1% up to 50%. When you consider the high value of the product, no losses from contamination are acceptable. Routine cleaning and aseptic procedures can decrease your losses to less than 1%. Laboratories should have easy to wash walls and floors. Acrylic or urethane epoxy wall paints Can be used; cement floors can be painted with an epoxy or urethane floor enamel or have an inlaid linoleum installed. High efficiency particulate air (HEPA) filters or regular furnace filters can be installed

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over air intakes to the laboratory or on furnaces. If possible, an enclosed entrance should precede the laboratory; sticky mats can be laid there to help collect dirt from the outside, or shoes can be removed. The traffic pattern and work flow in a laboratory must be considered in order to maximize cleanliness. The cleanest rooms or areas are the culture room, i.e. primary growth room, and the aseptic transfer area. It is best to design these rooms so they are not entered directly from the outside of a building. The media preparation area, glassware washing area, or storage area should be located outside these rooms. The primary growth room and aseptic transfer room should be enclosed with doors leading to each. Traffic through these areas can be minimized by installing pass-through windows. Ideally, the media preparation area would lead to the sterilization area, which would lead to the aseptic transfer  room and eventually the primary growth room. Unusual requirements for electricity and fire safety dictate that power  installation be done by professional electricians. Most wiring will require 110 volts. Temperature and fire alarms are to be connected directly to telephone lines to give fast warnings of problems. An emergency generator should be available to operate essential equipment during power outages.

GLASSWARE WASHING AND STORAGE AREA

The glassware washing area should be located near the sterilization and media preparation areas. When culture vessels are removed from the growth area, they are often autoclaved to kill contaminants or to soften semi-solid media. The vessels can be easily moved to the washing area if the autoclave or  pressure cooker is nearby. Locate the glassware storage area close to the wash area to expedite storage; these areas also need to be accessible to the media preparation area. The glassware area should be equipped with at least one large sink; two sinks are preferable. Adequate work space is required on both sides of the sink; this space will be used for glassware soaking tubs and drainage trays. Plastic netting can be placed on surfaces near the sink to reduce glassware breakage and enhance water drainage. The pipes leading from the sink can be PVC to resist damage from acids and alkalis. Both hot and cold water should be available with water distillation and/or deionization devices nearby. Mobile drying racks can be stored nearby and lined with cheesecloth to prevent water dripping and loss of small objects. Locate ovens or hot air cabinets (75 C) close to the glassware washing and storage area. Dust-proof cabinets, low enough to allow easy access, can be used in the storage area.

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MEDIA PREPARATION AND STERILIZATION AREA

The water source and glassware storage area should be convenient to the media preparation area. Benches, suitable for comfortable working while standing (34 to 36in.) and deep enough (24 in.) to hold equipment listed below are essential. Their tops should be made with molded plastic laminate surfaces that can tolerate frequent cleanings. There is a variety of equipment available for micropropagation laboratories; this equipment is generally located in the media preparation area. The equipment budget will determine the type and amount purchased. All laboratories need the following basics: 1. Refrigerator/freezer -- This is needed to store chemicals and stock solutions. Small laboratories may find it adequate to use countertop refrigerators. 2. High quality water --Bottled water can be purchased inexpensively and placed in the media preparation area. Larger businesses may find it economical to obtain distillation or deionization devices; these would normally be located in the glassware washing area. Small, inexpensive, low production Pyrex distillation devices can be purchased by small businesses that want the convenience of a still, but not the cost. 3. Balances  -- High quality balances are essential for a micropropagation laboratory; this is one area where it is difficult to find an inexpensive substitute. A triple beam balance is useful for large amounts over 10 grams, but a balance that can measure down to 2 mg is essential. Most laboratories have both a microbalance and a less sensitive top loading balance; the latter can be used more quickly and efficiently for less sensitive quantities. 4. Hot plate/stirrer --At least one hot plate with an automatic stirrer is needed to make semi-solid media. This purchase can be eliminated by using a stove and hand stirring the media while it heats; however, the time saved by using a stirring hot plate is worth the money spent. 5. pH meter --This is needed to measure media pH. Some laboratories use pH indicator paper, however this method is considerably less accurate and could severely affect the results. 6. Aspirator or vacuum pump --Aspirators can be easily attached to a water  source and used for filter sterilization of chemicals. They are also used to disinfest plant material. Vacuum pumps are faster and more efficient, but also more expensive. 7. Autoclave  -- An autoclave or pressure cooker is a vital part of a micropropagation laboratory. High pressure heat is needed to sterilize media,

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water, glassware, and utensils. Certain spores from fungi and bacteria will only be killed at a temperature of 121 F and 15 pounds per square inch (psi). Self  generating steam autoclaves are more dependable and faster to operate. 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.

PRIMARY GROWTH ROOM

Temperature, relative humidity, lighting units, and shelves need to be considered in the culture room. All of these environmental considerations will vary depending on the size of the growth room, its location, and the type of plants grown within it. For example, a small primary growth room located in a cool, North American climate, can be placed in an unheated or minimally heated basement. The ballasts from the fluorescent lights do not need to be separated; rather they can be used as a heating source. Excess heat can be blown out of  the growth room and used to heat other parts of the basement or building. In this case, solid wood shelves with air spaces located between shelves are recommended to prevent the cultures on shelves above lights from becoming over-heated. A larger growth room located in an above-ground location may need to have remote ballast and/or a heat pump installed. Shelves in a larger  growth room could then be glass or expanded metal. Temperature is the primary concern in culture rooms; it affects decisions on lights, relative humidity, and shelving. Generally, temperatures are kept 76 +/2 F. Heating can be accomplished by traditional heating systems supplemented with heat from light ballasts or space heaters. Cooling the room is usually a greater problem than heating; cooler temperatures can be obtained by installing heat pumps, air conditioners, or exhaust fans. Using outside windows to cool culture rooms invites contamination problems in the summer and humidity problems in the winter. Some plant cultures can be kept in complete darkness; however, most culture rooms are lighted at 1 klux (approximately 100ft-c) with some going up to 5 to 10 klux. The plant species being micropropagated will determine the intensity used. The developmental stage of the plants will also help determine if  wide spectrum or cool white fluorescent lights are used. Rooting has been shown to increase with far-red light; therefore, wide spectrum lights should be used during stage III and cool-white lights can be used during Stages I and II.  Automatic timers are needed to maintain desired photoperiods. Reflectors can be

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placed over bulbs to direct their light. Heat generated by the lights may cause condensation and temperature problems. In addition to using procedures previously mentioned, small fans with or without polyethylene tubes attached, can be placed at the ends of shelves to increase air flow and decrease heat accumulation. 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.

ASEPTIC TRANSFER AREA

In addition to the primary growth room, the aseptic transfer area needs to be as clean as possible. It is preferable to have a separate room for aseptic transfer; this decreases spore circulation and allows personnel to leave shoes outside the room. Special laboratory shoes and coats should be worn in this area. Laminar flow hoods or still-air boxes can be placed in this room and used for all aseptic work. Ultraviolet (UV) lights are sometimes installed in transfer  areas to disinfect the room; these lights should only be used when people and plant material are not in the room. Safety switches can be installed to shut off the UV lights when regular room lights are turned on. Surfaces inside the aseptic transfer area should be smooth to minimize the amount of dust that settles. Several electric outlets are to be installed to accommodate balances, flow hoods, bacti-cinerators, and microscopes.

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LABORATORY SET-UP AND CHECKLIST OF PTC REQUIREMENTS 1. Parts of a TC Laboratory a. Preparation Room b. Inoculating Room c. Culture Room d. Greenhouse/Nursery 2. List of Laboratory Equipment:: a. Air-conditioner k. Kitchen Stove b. Telephone* l. pH Meter   c. Triple Beam Balance d. Pressure Cooker/Autoclave e. Analytical Balance m. Computer * f. Heavy Duty Stove n. Printer * g. Blender / Osterizer o. Rotary Shaker  h. Refrigerator  i. Distilling apparatus  j. Laminar Flow Hood p. Fume Hood 3. Laboratory Furniture a. Working Table b. Filing Cabinet c. Hood Table d. Book Shelves e. Ladder f. Cabinets

g. Culture shelves h. Drying Racks i. Push Carts j. Chairs / Stools k. Office Table l. Shelves

4. Laboratory Glasswares / Plastic Wares a. Glass Beaker, 250, 500, 1000 ml b. Volumetric flask, 500, 1000 ml c. Brown Reagent Bottles 1000 ml d. Petri dishes e. Alcohol Lamp w/ wick f. Culture Bottles g. Erlenmeyer flasks 125, 250 ml h. Pippettes 10 ml. i. Graduate Cylinder   j. Glass dropper  k. Pipette tips

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5. Laboratory Utensils Pipette Pump / aspirator Pippetor Scooper Knife Scissors 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

Scalpel Forcep Scraper   Spatula Microspatula

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  

ii. Polypropylene plastic jj. Water drum. kk. Plastic Trays ll. Matches/Lighter   nn. Insecticidal spray oo.Pot Holder   pp.Light Starter   qq.Ballpen

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

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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 of sterilizing a volume of medium. Volume of media in container 20 to 50 ml 75 to 225 ml 250 to 500 ml 1000 ml 1500 ml 2000 ml

Time (Minutes) 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.

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MICROWAVE STERILIZATION (1991) Microwave sterilization is now done in some plant tissue culture media. However, this technique is not very reliable due to uneven heating. In microwave ovens, heat is caused by turning water molecules at 360 degrees turn, not directly to he at 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 dish’s surface is enough to sterilize it. Let it cool first before putting a plant to dissect since the dish surface is hot.

Effectiveness of some surface sterilizing agents Sterilizing agent

Concentration used

Duration (min) Effectiveness

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

 A comparison of the effectiveness and properties of common surface sterilants for explants Sterilizing  Agent

Concentration Used

Ease of removal

Treatment Time (min.)

Sodium hypochlorite Calcium hypochlorite Hydrogen peroxide Bromine water Silver nitrate Mercuric chloride  Antibiotics

1-1.4%a 9-10% 10-12% 1-2% 1% 0.01-1% 4-50 mg/l

+++ +++ +++++ +++ + + ++

5-30 5-30 5-15 2-10 5-30 2-10 30-60

Remarks

Very effective Very effective Effective Very effective Effective Satisfactory Effective

 A Common usage rate is 20% v/v of commercial solution Source: Yeoman & Maclead, 1977. NOTE: Use Mercuric chloride as a sterilant as a last resort. DO NOT dispose mercuric chloride into the sink as it is very toxic. Instead, contain the used sterilant solution in a sealed plastic  vessel.

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CHAPTER V BREEDING TECHNIQUES: POLLINATION & POLLEN STORAGE Orchid flowers need to be pollinated in order for it to form a fruit, which contain the seeds. Orchid flowers are usually pollinated 4 days after the flowers have opened, however some breeders has found out that orchid flowers could already be pollinate while still closed. Orchid flowers are hermaphrodites, meaning each flower have a male pollinia and a female stigma (see Parts of an Orchid Flower). Orchids are unique group of plants, having the male and female flower parts fused into a column. Also, all orchids share the same floral arrangement of having 3 sepals (1 dorsal, 2 lateral) and 3 petals (2 lateral, one modified into a lip or labellum). The pollinia is a mass of pollen grains, usually covered by an anther cap. Pollination is accomplished by placing the pollinia into the stigma of the flower. If the pollinia is placed into the stigma of the same flower or of the same plant, this is called self-pollination. However, if the pollinia is placed into the stigma of another orchid species/cultivar, then it is called 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.

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POD HARVESTING Pods are harvested once they are mature and or have burst. This means that the pollen tube of the pollen have reached the ovules and have fertilized it. The grower has the option of  selecting the technique in which he could apply to sow his orchid seeds. Thus this would affect the schedule of his pod harvesting. There are two methods of sowing seeds in orchids. They are the dry pod culture and the green pod culture. The dry pod culture is also called seed culture since the explant to be used are the mature viable seeds. In this method, the orchid capsule have already burst, and the seeds are ready to be carried by the wind. Seeds are powdery or dust-like, and are white, yellow, or brown in color. It takes a long time for the orchid capsule to mature (4-10 months), and usually, as it opens, some of the seeds are already lost. Thus, it is very important that orchid capsules be checked regularly for signs of maturity (yellowing and presence of cracks). Once the capsule is ready it is cut off from the flower stalk and wrapped in dry tissue paper and brought to the laboratory. Seeds in the capsule have to be properly dried in a dessicator. A dessicator is usually composed of a large bottle with lid and with a lining of metal screen at the bottom. Beneath the metal screen, is the desiccant pockets which could be obtained from drug stores. The desiccant pocket is composed of chemical salts (silica gel or calcium chloride) which absorb moisture from the air inside the bottle. Once dried, the seed could also be stored for some time in the refrigerator or immediately sown in flasks. In green pod culture, also called embryo culture or embryo rescue, the immature seeds are the once used as explant. In orchids, the interval from pollination to fertilization varies from one genera to another and varies from 10 days to 6 months. Studies have shown that orchid embryoes become viable and are capable of germination soon after fertilization. This takes place long before fruit maturity. Green pod culture offers the advantage of shortening the time from pollination to flasking. Below is a table showing the time (in days) when immature capsules from the following genera are ready for flasking

Table 1. Number of days when seed pods of certain genera will be ready for green pod culture. (Also depends on species and cultivar) 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

c. Potassium phosphate monobasic KH2PO4

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

2. Dissolve the following salts in 900 ml distilled water one at a time. 3. Bring the volume to 1000 ml in a volumetric flask using distilled water. 4. If either one of the crystals does not dissolve, partially heat the solution in the hot-plate stirrer  until fully dissolved. The solution will be clear-light yellow in color. 5. Place and store stock solution in a tightly covered brown bottle inside the refrigerator. 6. Label the bottle with the corresponding chemical name, date prepared and amount to use per  liter media.

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Note: Sodium EDTA is used as chelator for Ferrous sulfate, to prevent it from precipitating. Ferrous sulfate will not likely to dissolve without sodium EDTA. Use this stock at 10 ml each per  liter of stock solution.

Preparation of Standard Solution for Adjusting pH of Medium (KOH and HCl). To prepare 1 liter of 1 Normality Potassium Hydroxide (KOH) or Sodium Hydroxide (NaOH): 1. Dissolve 56.0 g KOH (or 40.0 g. NaOH pellets) in approximately 900 ml distilled water. This is an exothermic reaction, wherein the solution will be a little bit warmer than before. 2. Cool the solution to room temperature. 3. Dilute or bring volume to 1 liter in a volumetric flask using distilled water. 4. Transfer to brown glass bottle and label. NOTE: 1 N KOH is used when the nutrient medium has a low pH (pH below 5). Add 1 N KOH drop by drop until the desired pH (5-6) is attained. Be careful with the said chemicals, they are corrosive. To prepare 1 Normality Hydrochloric Acid (HCl). 1. Add 60 ml concentrated HCl (analytical reagent) to approximately 500 ml distilled water in a volumetric flask drop by drop (Be careful, the solution is corrosive). 2. Mix by stirring briskly. 3. Dilute or bring the volume to 1 liter by adding distilled water. 4. Transfer to brown glass bottle and label. NOTE: 1 N HCl is used when the nutrient medium has a pH higher than pH of 5.6. 1 N HCl is added drop by drop until the desired pH is attained. In pipetting out the pure HCl for dilution, do this inside a fume hood. Be careful with the said chemicals, they are corrosive.

VITAMIN STOCK SOLUTION (Optional) Vitamins usually enhances growth of cultures in vitro. Vitamins used in orchid embryo culture includes Vitamin B complexes which help in the cells' metabolism. Vitamin B complex from drug stores can also be used. The vitamin stock solution below is that of Nitsch & Nitsch. 1.. Weigh the following and dissolve in 300 ml distilled water. Nicotinic acid 250 milligrams Pyridoxine HCl 25 milligrams Thiamine HCl 25 milligrams Folic acid 25 milligrams Biotin 2.5 milligrams Glycine 100 milligrams 2. Add enough distilled water to make a 500 ml solution in a volumetric flask. Folic acid will not usually dissolve. 3. Adjust pH to 7.00 to dissolve Folic acid by adding drops of 1 N KOH or NaOH. A clear  transparent solution will result. 4. Store vitamin stock in a brown bottle and inside the refrigerator. NOTE: Use 10 mL of this stock to make 1 Liter media.

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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.

PREPARING THE KNUDSON C MEDIUM To Prepare 1 Liter Knudson C Germinating Media, the following are needed: Knudson C Basal Stock Solution 50 mL each Iron-EDTA Stock 10 mL Vitamin Stock (Optional) 10 mL Myo-inositol stock (Optional) 10 mL Coconut Water (from green coconut) 100 mL Tomato Puree (from 3 tomatoes) 150 mL Sucrose (Table sugar) 20 grams  Agar 8 grams Distilled water Enough to make 1 L media Procedure: 1. Strain / Filter the coconut water (not coconut milk) using cheese cloth or cotton placed in a plastic or glass funnel to remore floating impurities. Get 100 ml of this and place in a 1000 ml beaker. Excess coconut water is stored by placing it in a plastic container and stored in freezer. 2. Pour 50 mL each of the Knudson C stock into the beaker. 3. Pour 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 3 medium size tomatoes in a blender/osterizer and add 50 ml distilled water. Osterize the tomatoes until a puree is produced. Strain the seeds and add the content into the media. 7. Weigh the agar and place in into a separate container. It is mixed with about 100 ml distilled water in a glass (Pyrex) beaker or a small sauce pan. The mixture is made to boil in a magnetic hotplate stirrer or a stove until the agar is cooked. The agar mixture is then added to the media. 8. Enough water is added up to the 1 L mark. The mixture is mixed thoroughly. 9. The pH is adjusted to 5.6 (adding HCl or KOH) using a pH paper or pH meter. 10. The media is then dispensed into catsup bottles or Erlenmeyer flasks. 11. Seal the bottles with metal or plastic caps. Wrap the caps with paper sheets by using rubber  bands. 12. Sterilize the bottles in a pressure cooker at 15 psi for 30 minutes. 13. Place sterilized culture bottles in the culture room to cool.

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PREPARING THE KNUDSON C REFLASKING MEDIUM To Prepare 1 Liter Knudson C Reflasking Media, the following are needed: Knudson C Basal Stock Solution Sucrose (Table sugar) Coconut Water (from green coconut) Banana Homogenate  Agar  Activated Carbon Vitamin Stock (Optional) Iron-EDTA Stock Myo-Inositol Stock (Optional) Distilled water

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

 A. To Prepare 1 Liter Knudson C Initiating Media, the following are needed:: Knudson C Basal Stock Solution Coconut Water (from green coconut) BA Stock Distilled water

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.

27

5. Inside the laminar flow hood or sterile chamber, sterilize the shoot with 1% calcium hypochlorite or 20% bleach with Tween 20 for 30 minutes. 6. In a sterile petri dish, rinse three times with sterile distilled water. 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)

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PROCEDURE FOR IN VITRO PROPAGATION OF VANDA I. INITIAL CULTURE (Establishment of aseptic culture)

 A. To Prepare 1 Liter Knudson C Initiating Media, the following are needed: Knudson C Basal Stock Solution Coconut Water (from green coconut) BA Stock Kinetin Stock Distilled water

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.

Selection, Sterilization and Explant Isolation

1. Select and cut a young inflorescence with undifferentiated buds not more than 5 cm. Long 2.Wash well with detergent and tap water. 3. Rinse three times with sterile distilled water. 4. Inside the laminar flow hood or sterile chamber, sterilize the flower bud with 20-50% bleach with Tween 20 for 30 minutes.

29

5. Rinse three times with sterile distilled water. 6. Subject the flower bud in 20 % v/v hydrogen peroxide for 30 minutes. 7. Rinse three times with sterile distilled water. 8. Remove outer bracts surrounding the bud in sterile petri dish. 9. Cut young flower bud into 0.5 to 1 cm segments. 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 + 1 ppm Kinetin + 7 grams agar (per Liter).

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 + 50 ml coconut water + 50 g. saba + 10 g. sugar  + 7 g. agar (per liter), then transfer to Knudson C + 100 ml coconut water + 100 g. saba + 20 g. sugar  + 7 g. agar + 1 g. activated charcoal + 10 ppm IBA (per liter)

30

PROCEDURE FOR FLOWER STALK PROPAGATION PHALAENOPSIS  I. INITIAL CULTURE (Establishment of aseptic culture)

 A. To Prepare 1 Liter Knudson C Initiating Media, the following are needed: Knudson C Basal Stock Solution Coconut Water (from green coconut) Sugar NAA Stock Distilled water

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

o

C.

9. Incline sterilized test tubes in the culture room to cool.

Selection, Sterilization and Explant Isolation

1. Select a mature flower spike whose flower has just wilted. 2. Wash the stalk with detergent and water  3. Wipe the stalks with cotton wet with 95% ethyl alcohol 2-3 times 4. Cut the stalk into sections from 1 cm to 2 cm length. Each cutting must have a node.

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5. Soak the cuttings in 20% bleach with Tween 20 solution for 20 minutes. 6. Wash the stalk with sterile distilled water three times 7. Remove the outer scales / bracts. 8. Dip cuttings in 5% bleach solution for 10 minutes and dip in sterile distilled water for 2 to 3 minutes. 9. Dip the portion of cuttings in a test tube with the culture medium. 10. Place in the culture room. Wait for emergence of plantlets.

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 + 1 ppm Kinetin + 7 grams agar   (per Liter). 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 + 50 ml coconut water + 50 g. saba + 10 g. sugar  + 7 g. agar (per liter), then transfer to Knudson C + 100 ml coconut water + 100 g. saba + 20 g. sugar  + 7 g. agar + 1 g. activated charcoal + 10 ppm IBA (per liter)

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MICROPROPAGATION OF CATTLEYA AND ALLIES.

Explant: Axillary buds or meristems from 2 to 5 cm shoots. Treatment: 1. Wash the shoot in tap water and detergent. Dip the shoot in ethyl alcohol for  10 seconds. Mix in 10% bleach for 15 minutes. Rinse briefly in sterile distilled water and dry on sterile petri dish.

2. Under a dissecting microscope, remove carefully the overlapping leaves. 3. To help prevent browning, dissect under fresh sterile antioxidant solution. Watch for buds about 2 mm in size at the base of the leaves. 4. Excise the Whole buds, severing just below the point of attachment. These may be cultured, or dissected further to obtain meristem with one or two pairs of  leaf primordia. 5. Media: Grow buds or meristems in medium with 100 ml/l coconut water. Best started in agitated liquid medium but agar solidified medium is often satisfactory and required for stage III. Knudson C + MS micro-stock + 10% coconut water + 2% sucrose and 0.6 agar  pH = 5.5 Stage I & II without agar; Stage III w/ agar  Light: continous light at 100-300 footcandles from cool white fluorescent lamps. Temperature:: 26 oC Discussion: Cattleyas multiply better in liquid than on agar, at least initially. The theoretical reason is that the agitation inhibits polarity (orientation). Once polarity is established, the cultures put out shoots and roots and mature. The initial growth that is desired for multiplication is a mass of protocorms. As soon as this mass grows to 1 cm it should be divided and put back into liquid or agar medium.

When the culture is on agar frequent division will help upset orientation and delay plantlet formation..

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MICROPROPAGATION OF CYMBIDIUM  SPECIES & HYBRIDS.

Explant: apical meristem Treatment:  Remove outer leaves from 3 cm shoots. Dip in ethyl alcohol for 2 seconds then mix in 10% bleach for 15 minutes. Rinse in sterile distilled water. Remove remaining leaves. Excise meristem consisting of apical dome, two leaf  primordia, and a cube of tissue, all less than 0.5 mm. Media: liquid Modified Knudson C or Vacin and Went

 After 3-4 subcultures, transfer to media with agar (KC or Morel and Muller) for  shoot and root development. Light: continous light for 100 fc from fluorescent lights Temperature: 22 oC

 Apical meristems in culture follow similar development to orchids seeds wherein they produce protocorms, bulbous tissues with root hairs.

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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Green Pod Culture Technique: 1. Mature orchid pod is washed with detergent (e.g. Teepol) and running water. Inspect pod for  holes, insect damage or rotting. 2. Inside the transfer chamber of laminar flow, the pod, scalpel and forcep is sterilized by dipping in 95 % ethyl alcohol and flaming at least 3 times over an alcohol lamp. 3. Hands are washed with soap and water, and then wiped with 70% isoprophyl alcohol. At this stage, all manipulation is done inside the laminar flow. 4. In a sterile Petri dish, the pod is placed and cut crosswise or lengthwise (depending on size of  pod). 5. The ovules (white, yellow or light yellow-brown in color) are scrapped off inoculated into the germinating media. 6. The bottles are labeled (date and cultivar/species). Accession numbers can be used. 7. The bottles are placed in lighted culture shelves. After one week, contaminated cultures are removed and sterilized (decontaminated) with procedure similar to sterilizing fresh culture media. 8. After 3 weeks, viable ovules will show signs of germination (enlargement of ovules and greening). After 2 months or when protocorms are 1 cm long, cultures are ready for reflasking.

Dry Pod Culture In this process, dried orchid seeds (orchid capsules are open) are used. This process is usually injurious to orchid seeds and less seeds germinate compared to green pod culture. 1. Place the pod over a clean sheet of white paper. 2. Tap the pod over the paper to release the dust-like seeds. 3. Place seeds into a vial or test tube with cap. 4. Mix 1-2 grams sugar in 100 ml water. Then place the solution into the vial or test tube with seeds and add a 1-2 drops Tween 40 or detergent (it serves as a surfactant). Cap and shake the test tube until the seeds are submerged or fully wet. Let the seeds in the solution for 16-24 hours. This technique will germinate any bacterial or fungal spores in the seed and it will make them susceptible to the sterilizing solution afterwards. Some Fungal/Bacterial spores are usually resistant to the sterilizing solution. 5. After 24 hours, pour out/remove the sugar solution, and leave the seeds inside. Pour 10% Chlorox solution plus 1-2 drops Tween 80 into the vial. Cap and shake vigorously and let it stay there for 10 minutes. Let seeds settle down. (A centrifuge could be very helpful in settling seeds down in the bottom of the test tube) 6. Wash the seeds 3 times with sterile distilled water. 7. At the last washing, leave a little sterile distilled water, and pour the seed and water mixture into the germinating media. 1 or half a drop of the solution with seeds is enough for each flask.

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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.

Orchid Dry Pod Culture Using Filter Paper Wrap This procedure is provided by Dr. Lilian Pateña of the Institute of Plant Breeding. The technique is rather time consuming, however, it solves the problem of the floating seeds usually associated with the technique using a test tube or vial. 1. Obtain or collect dry orchid seeds in a bottle or piece of paper. 2. Cut 2 inches X 2 inches filter paper squares (it need not be sterile). The fold the filter paper  squares in half lengthwise or cross wise. 3. Place a pinch (about 1/10 of a teaspoon or less) or orchid seeds in the middle of the filter paper  square. 4. Fold the filter paper square in half, enveloping the seeds. Fold the sides and top of the folded filter paper square 2x to seal the enveloped seeds. 5. Fasten the two tips of the envelope using a stapler. Do not puncture a hole into the envelope,  just fasten the topmost right and left corner of the envelope with the staple wire. 6. Inside a laminar flow hood, place the filter paper envelope in a sterile petri dish. Pour the sterilizing solution (20% solution of commercial bleach and water plus some drops of Tween 20) in it. Pour enough to cover the filter paper envelope, about half the level of the petri dish. Slightly agitate the petri dish to mix the solution with the envelope. Do this for 15 to 20 minutes. The orchid seeds will now slightly change its color, as being bleached by the chlorox solution. 7. After 15 - 20 minutes, decant the sterilizing solution and wash the filter paper envelope in sterile distilled water 3x in the same Petri dish. Use sterile forcep in moving the filter paper  envelope. 8. Allow the filter paper envelop with the seeds to dry up in the air flow inside the laminar flow hood for about 4 hours. Let it stay in the petri dish and place a glass cover slightly open to allow the sterile air from the laminar flow hood to enter the Petri dish. 9. When the filter paper envelope is completely dry, remove the staple wires to open the seal of  the envelope using a sterile forcep and scalpel. A sterile surgical scissors can also be used to cut the paper open. 10. Carefully hold the filter paper envelop over an opened culture bottle, using a sterile forcep, and gently tap the envelope to release the powder like-orchid seeds into the flask containing orchid germinating media.. 11. Cap the bottle and label. Place the bottle in a lighted shelf inside the culture room. The orchid seeds will germinate after 3 weeks. Discard any contaminated cultures. 12. Subculture the orchid protocorms after 2 months into flasks containing fresh reflasking media.

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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.

COMPOTTING (Community Potting). 1.

Using a forcep or spoon-like tool, the plantlets are scooped out of the bottles. Care must be observe so that roots would not be damaged. (For others, cheap glass bottles like catsup or  Gilbeys bottles, are wrapped with newspaper or masking tape, and cracked open using a hammer in order to ease the retrieval of plants. 2. Place the plantlets into a basin of water to easily remove and clean the clinging agar media from the roots. (It is needed to completely remove the agar media or else it will attract fungal diseases and ants.) 3.

The plantlets are then dipped in a dilute solution of fungicide (either Dithane or Captan) and with a few drops of Rooting Hormone (Root Booster, Hormex or others) for 3 minutes.

4.

The plantlets are then sorted out base on size and placed on a sheet of old news paper to dry.

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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,

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Paphiopedilum spp., Peristeria imschootiana & Vanda coerulea

elata,

Phragmipedium

spp.,

Renanthera

The Orchid Species Group was set up in August 1984 following the 11 th 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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Mass Propagation of Orchid Species.  Embryo culture is a powerful tool in mass producing orchids. It is a fact that orchids produce thousands to millions of dust-like seeds per plant. If enough orchid plants could be pollinated and produce a capsule, the seeds could be grown in designated plant tissue culture laboratories and could be later be used to re-stock the forest or the ones used for  trade. With this, the habitat where the orchid species are collected will not be touched.. Priorities could be given to endangered and threatened species and also to those highly demanded for trade. Knowledge with breeding and genetics is a requirement in this in order to prevent producing weak and inferior plants.  Also, learn how to vegetatively divide your plants. Both private and government sectors could be tapped for this. In collecting specimens from the forests, do not get all the plants.  As much as possible, be responsible enough to get only the seedlings, and leave enough matured plants that could survive and reproduce for the next generation. Protect natural habitats of orchids.   The main problem of orchid conservation is habitat destruction. Let us support in the creation of national parks and nature reserves. Salvage plants from damaged or threatened areas. Do travel in areas where there are forest clearing to give way to road building, logging, dam building, agricultural development, mining, etc. and save these orchids from being killed. Establish rescue centers for salvaged or confiscated plants. Do create centers in coordination with the local orchid society and the government which will care for salvaged or confiscated plants, and where the plants could be used for education, propagation and conservation purposes. The center needs to be well equipped with physical facilities (like a nursery, greenhouse, a laboratory) and technical expertise. Help create new habitats from damaged areas, including urban environments.  This could be in the form of small parks or conservatories, wherein a small patch of land could be protected for the growth of its native orchid species and other plants. Within this new habitats, orchid species which are found naturally, could then be reintroduced. These sites could later be used for eco-tourism. Educate the amateur collectors to collect only for their own and to follow a certain code of collecting plants.   Amateur collectors need to be trained not to collect indiscriminately, from the wild, and always follow the code of  orchid conservation ethics. Collect only a number of plants per each species which you can take care off. As much as possible, collect only seedlings and

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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 

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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.

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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.

Main Orchid Types Traded in the Country. 1. Vanda 2. Renanthera

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3. Phalaenopsis & Doritis 4. Dendrobium 5. Cattleya group 6. Paphiopedilum 7. Spathoglottis 8. Grammatophyllum 9. Cymbidiums 10. Oncidiums 11. Orchid Species & Botanicals

The Need to Master the Cultural Requirements of Orchid

For the orchid grower or trader, one needs to understand the orchids’ cultural requirement in order for the plants to grow healthy at and its best appearance. These are as follows:  Light.  The plant will prefer a slightly brighter location to full sun. Light is the most important element in the successful production of orchids. Based on the type of orchid, the plant will prefer exposure to morning sun and could tolerate direct sun, but must be protected from it during very hot months. Sunlight can be filtered using 2-3 layers of net 8 feet above the plants during the summer period.  Altitude is also a factor which influence light intensity.

 As a general rule, terete Vanda, Spathoglottis and Oncidium can thrive on direct sun. On the otherhand, Dendrobiums, Semi-Terete / Strapleaf Vandas and Cattleyas (including their allies) are better placed in a net-house or greenhouse with 75-60% light, while Phalaenopsis and some terrestrial orchids are better  placed in 50% shade nethouses or greenhouses. Watering. Orchid plants prefer and tolerates a little bit drier condition. Due to the plants’ anatomical and physiological structure, the orchid can be watered every other day or even less, like once every other day (e.g. Vandas and most monopodial orchids), and once every third day or once a week (sympodial orchids), just as long as the surroundings and companion plants are kept moist to provide high humidity. However, as a grower, one has to look at how the plants reacts to watering, and thus, needs to be adjusted accordingly. If the plants are becoming dehydrated due to intense heat, it may be much proper to water them 3-4 times a day. Plants needs to be grouped together based on watering requirements. Moreover, these plants need to be protected from excessive monsoon rains through the establishment of a plastic-covered greenhouse, as prolonged moisture can cause rotting and attract pests and diseases. Ventilation. Orchids prefers an area with slight breeze but not a very windy area, in order to dry some of the moisture in its foliage and potting media

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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..

In the commercial production of orchids, diluted Fertilization. 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.

Different Approaches in the Orchid Business Commercial Orchid Trader   – these group buys and sells plants locally, or may also export and import plants. They don’t necessarily have a farm or a store, however, they do have a lot of connections and linkages. They usually buy wholesale then distributes them on a retail basis. They usually have plant stores strategically positioned in garden centers in major cities. They are concentrated on the business aspect of orchids.

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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.

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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  th Orchids. Philippine Journal of Crop Science 34(S1). Poster Paper presented in the 13  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,.

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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 Baños, 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.

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PALMER, T. 2001. “Dendrobiums of the Philippines: Traditional Uses.” In: nd Orchids of the Philippines (22  Annual Orchid Workshop, August 4, 2004) Houston, Texas, USA: The Houston Orchid Society. pp.47 PAPTC. 2002. The Directory of Tissue Culture Laboratories in the Philippines. ed. Zamora, A.B. . Laguna, Philippines: Philippine Association for Plant Tissue Culture. c/o Institute of Plant Breeding, College of Agriculture, University of the Philippines Los Baños. PAPTC. 2002. (Souvenir Program) Convention Year 2002: "Enhancing Plant Tissue Culture and Entrepreneurship in the Philippines". Laguna, Philippines: Philippine Association for Plant Tissue Culture. c/o Institute of Plant Breeding, College of Agriculture, University of the Philippines Los Baños,. PCARRD. 1994. The Philippines Recommends for Orchids. rev. ed. Los Baños, Laguna. Philippine Council for Agriculture, Forestry and Natural Resources and Development Department of Science and Technology QUILANG, A.B.; BAUTISTA, N.R.; TAYLAN, G.B.; and MADERA, R.F.2001. "Comparative Study of the Germination Performance of Dendrobium 'Lunsom Green' (Orchidaceae) in Murashige & Skoog's, Vacin & Went and Knudson C Media". Quest. (Vol II, p.7 ) Mandaluyong City, Philippines: Rizal Technological University. QUILANG, A.B.; BAUTISTA, N.R., TAYLAN, G.B.; MADERA, R.F.; and PULMA, C.C. 2001. Growth Response of Dendrobium crumenatum (Orchidaceae) to Knudson C Media with Supplements. Quest. (Vol II, p.11 ). Mandaluyong City, Philippines: Rizal Technological University QUILANG, A.B.; BAUTISTA, N.R.; TAYLAN, G.B.; & MADERA, R.F. 2001. "Growth and Flowering Performance of Dendrobium cv. 'Anching Lubag X  Allan Umaki' (Orchidaceae) to Foliar Fertilizer." Quest. (Vol II, p.5 ). Mandaluyong City, Philippines: Rizal Technological University. QUILANG, A.B.: BAUTISTA, N. R.; TAYLAN, G. B.; MADERA, R.F.; and PULMA C.C. 2001."Growth of Dendrobium ternatense (Orchidaceae) in Three Different Culture Media: Knudson C., Murashige & Skoog, and Vacin & Went". Quest. (Vol II, p.10). Mandaluyong City, Philippines: Rizal Technological University, QUILANG, A.B.; BAUTISTA, N. R.; TAYLAN, G. B.; MADERA, R.F.; and PULMA, C.C. 2001."Growth & Embryo Culture of Grammatophyllum sp. in Three Culture Media: Knudson C, Murashige & Skoog, and Vacin & Went". Quest. (Vol II, p.8). Mandaluyong City, Philippines: Rizal Technological University, ROBERTS, JACQUELINE A. et al. 1995. CITES Orchid Checklist Volume 1 & 2. UK. Royal Botanic Gardens, Kew. STEWART. JOYCE . 1987. "Orchid Conservation: Survival and Maintenance of  Genetic Diversity of All Orchids Throughout the World." American Orchid Society Bulletin (August 1987). pp. 822-827. STEWART. JOYCE. 1986. "Orchid Conservation at the International Level."  American Orchid Society Bulletin (March 1986). pp. 242-246.

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STEWART. JOYCE. Ed. 1992. Orchids at Kew. Singapore: HMSO Publications Centre. UPLB. 1999. Orchid Micropropagation. (unpublished hand-out). Ornamental Crops Division, Dept. of Horticulture, Laguna, Philippines: University of the Philippines at Los Banos. VALMAYOR, H. L. 1984. Orchidiana Philippiniana. Vol. 1. Philippines: Eugenio Lopez Foundation, Inc.

APPENDIX  APPENDIX A

PREPARATION OF R MEDIA FOR ORCHID EMBRYO CULTURE The R Medium was developed by the group of Dr. Ramon C. Barba and Dr. Lilian F. Patena, Institute of Plant Breeding, University of the Philippines at Los Banos, Laguna, Philippines.

METHODOLOGY  I. Macroelement Stock 20X Concentration (RM Macro) 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.

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III. Microelements Stock 200 X Concentration (RM Micro) 1. Weigh the following chemical reagents: a. Manganese Sulfate MnSO4 . H2O 0.13 grams b. Boric Acid H3BO3 0.020 grams c. Zinc Sulfate ZnSO4. H2O 0.020 grams d. Copper Sulfate CuSO4 . 5H2O 0.004 grams 2. Dissolve the following chemical reagents one at a time in a glass beaker with 700 ml distilled water using a magnetic stirrer. 3. When all the salts are dissolve, pour enough distilled water to make a 1000 ml solution. Pour  in a brown bottle, and label(Name of solution, Date Prepared). Store stock solution in the refrigerator. Note: Use 5 ml of this stock in making 1 L medium.

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.

V. Myoinositol Stock (Optional) 1. Weigh the following chemical reagents: a. Myo-Inositol 5.00 grams b. Ascorbic acid 1.25 grams c. Arginine 2.50 grams d. Tyrosine 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.

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VI. Ferrous Sesquestrene Stock  1. Weigh 1.25 grams Ferrous Sesquestrene and dissolve in a beaker with 200 ml distilled water  using a magnetic stirrer. 2. When all the salts are dissolve, pour enough distilled water to make a 250 ml solution. 3. Pour in a brown bottle, and label (Name of solution, Date Prepared). Store stock solution in the refrigerator. Note: Use 5 ml of this stock in making 1 L medium.

TO MAKE 1 LITER of R MEDIA 1. Measure the following solutions: a. RM Macro Stock b. Magnesium sulfate stock c. RM Micro Stock d. RM Vitamins Stock e. Myo-Inositol Stock f. Ferrous Sesquestrene Stock g. Sucrose h. Agar i. Coconut water (Buko type)  j. Yeast Extract k. Tomato Puree l. Banana Homogenate (Bungulan)

50 ml 50 ml 5 ml 5 ml 5 ml 5 ml 20 grams 8 grams 100 ml 1 gram 10 grams 50 grams

2. Combine solutions from a to l except in h = Agar. 3. In 1 L beaker, place 600 ml distilled water and Agar. Stir and Heat mixture in a stirring hot plate until it boils. Remove it from the hot plate when the agar has completely melted and combine it with the rest of the solution. 4. Adjust pH to 5.6 to 5.7 using either 1 Normality HCl or NaOH / KOH. 5. Dispense the mixture in individual culture jars (catsup or mayonnaise bottle). Cap bottles with plastic or metal caps, then wrap cap with used bond papers and rubberbands. 6. Sterilize in the pressure cooker at 15 psi for 30 minutes. Then place the culture media in the culture room to cool. Use it after 1 week. 7. Note: R Media is brownish red in color when sterilized with its distinctive odor.

REFERENCES: BARBA, RAMON C. & PATENA, LILIAN F.  1999. R Meidum, A New Medim for Tissue Culture of Orchids. Philippine Journal of Crop Science 34(S1). Poster Paper presented in the 13th FCSSP Annual Science Conference, Family Country Homes, General Santos City. ---------* If NH4H2PO4 is not available, use (NH 4)2HPO4.

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APPENDIX B

PREPARATION OF VACIN & WENT MEDIA The original composition of the Vacin & Went Medium is as follows (Vacin & Went, 1949): Composition Potassium nitrate  Ammonium sulfate Tricalcium phosphate Magnesium sulfate Potassium phosphate monobasic Ferric Tartrate Manganese sulfate Sucrose  Agar

Chemical Symbol KNO3 . NH4(NO3)2  4H2O Ca3(PO4)2 MgSO4 . 7H2O KH2PO4

Amount (g/L) 0.525 0.500 0.200 0.250 0.250

.

e2(C4H4O6)3  2H2O MnSO4 . 4H2O

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.

Preparation of Vacin & Went Stock Solution (20X). 1. Weigh the following chemical salts: Potassium nitrate  Ammonium sulfate Tricalcium phosphate Magnesium sulfate Potassium phosphate monobasic Ferric Tartrate Manganese sulfate

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.

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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

2. Dissolve the following salts in 900 ml distilled water one at a time. 3. Bring the volume to 1000 ml in a volumetric flask using distilled water. 4. If either one of the crystals does not dissolve, partially heat the solution in the hot-plate stirrer  until fully dissolved. The solution will be clear-light yellow in color. 5. Place and store stock solution in a tightly covered brown bottle inside the refrigerator. 6. Label the bottle with the corresponding chemical name, date prepared and amount to use per  liter media. 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. To prepare 1 Liter of Vacin & Went Medium, mix the following: Vacin & Went Stock Solution: Potassium nitrate  Ammonium sulfate Tricalcium phosphate Magnesium sulfate Potassium phosphate monobasic Manganese sulfate Ferric Tartrate (If there is a problem with Ferric Tartrate, it  can be replaced with Ferrous sulfate + EDTA = 10 mL ) Sucrose  Agar (Dissolve previously in boiling water)  Add enough distilled water to make 1 liter. Adjust pH to 5.6

50 mL 50 mL 50 mL 50 mL 50 mL 50 mL 50 mL

20.00 16.00

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 APPENDIX C. MURASHIGE AND SKOOG’S 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 ethylene  diaminetetra-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

Preparing an MS Stock Solution. Major Salts (MS Macro – 10 x) 1. Weigh the following salts Potassium nitrate  Ammonium nitrate Calcium chloride Magnesium sulfate Potassium phosphate   monobasic

Chemical Symbol KNO3 NH4NO3 CaCl2 . 2H2O MgSO4 . H2O KH2PO4

Grams 19.0 16.5 4.4 3.7 1.7

2. In a 1000 mL glass beaker, pour 700 mL of distilled water  3. Dissolve the salts above one at a time in distilled water, stirring each time briskly. Once all are dissolved, add enough distilled water to the 1000 mL   mark. 4. Store the stock solution in a brown bottle. Place label and the date the stock was made. Store in the refrigerator. This stock solution is 10 times the formula concentration. Use 100 mL of this stock in making 1 liter MS medium.

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Minor Salts (MS Micro – 100 x) 1. Weigh the following chemical salts: Manganese sulfate Zinc sulfate Boric acid Potassium iodide Sodium molybdate Copper sulfate Cobalt chloride

2. 3.

4. 5.

MnSO4 . 4H2O ZnSO4 . 7H2O H3BO3 KI Na2MoO4 . 2H2O CuSO4 . 5H2O CoCl2 . 6H2O

Milligrams (mg) 1680 860 620 83 25 2.5 2.5

Dissolve the first 5 chemical salts in 700 mL distilled water, one at at time in a mixing flask. 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). Add enough distilled water to make 1000 mL. This final solution is 100 times the formula concentration. 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

2. Dissolve the following salts in 900 ml distilled water one at a time. 3. Bring the volume to 1000 ml in a volumetric flask using distilled water. 4. If either one of the crystals does not dissolve, partially heat the solution in the hot-plate stirrer  until fully dissolved. The solution will be clear-light yellow in color. 5. Place and store stock solution in a tightly covered brown bottle inside the refrigerator. 6. Label the bottle with the corresponding chemical name, date prepared and amount to use per  liter media. Note: Sodium EDTA is used as chelator for Ferrous sulfate, to prevent it from precipitating. Ferrous sulfate will not likely to dissolve without sodium EDTA. Use this stock at 10 ml each per  liter of stock solution. Sodium ethylenediaminetetra-acetate Ferrous sulfate

Na2EDTA . FeSO4  7H20

0.0373 0.0278

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VITAMIN STOCK SOLUTION (Optional) (Nitsch & Nitsch, 1969) Vitamins usually enhances growth of cultures in vitro. Vitamins used in orchid embryo culture includes Vitamin B complexes which help in the cells' metabolism. Vitamin B complex from drug stores can also be used. The vitamin stock solution below is that of Nitsch & Nitsch. 1.. Weigh the following and dissolve in 300 ml distilled water. Nicotinic acid 250 milligrams Pyridoxine HCl 25 milligrams Thiamine HCl 25 milligrams Folic acid 25 milligrams Biotin 2.5 milligrams Glycine 100 milligrams 2. Add enough distilled water to make a 500 ml solution in a volumetric flask. Folic acid will not usually dissolve. 3. Adjust pH to 7.00 to dissolve Folic acid by adding drops of 1 N KOH or NaOH. A clear  transparent solution will result. 4. Store vitamin stock in a brown bottle and inside the refrigerator. NOTE: Use 10 mL of this stock to make 1 Liter media.

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.

Preparing the MS Medium 1.

To make a 1 Liter medium, measure the following stock solutions MS Macro Stock MS Micro Stock Fe-EDTA Stock Vitamins Stock Myo-Inositol Stock Coconut water Sucrose  Agar

2. 3. 4. 5. 6.

10 mL 100 mL 10 mL 10 mL 10 mL 100 mL 30 grams 10 grams

Combine the first 7 components in a glass beaker. Mix them briskly to dissolve the sucrose. Cook and dissolve Agar in 300 mL boiling water. Then add to the rest of the medium. Add enough distilled water to make a 1 Liter medium Adjust pH to 5.6 using 1 N HCl or 1 N KOH. Dispense medium into culture flask, cover and sterilize in pressure cooker.

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 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.

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 APPEDIX E PREPARING YAMADA’S MEDIUM This is a very simple orchid medium made from an orchid fertilizer, which has made orchid seed sowing simple and attractive to orchid hobbyists. Modified Yamada’s 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:

1. Weigh and measure all chemicals. 2. In a glass or plastic breaker, fill it with 500mL water, the drop one chemical (except the agar) at a time while continuously mixing it with a glass stirrer. 3. In a separate metal kettle or saucepan, mix 400 mL water and the Agar and sugar. Place the kettle or saucepan in an open flame and stirr occassionally until the water boils and the agar dissolves. Then pour the mixture into the beaker  with the other chemicals. 4. Add the coconut waer, osterized tomatoes and enough distilled water to make 1000 mL. 5. Test the pH to 5.6 using a pH paper and adjust the pH with 1N HCl or 1N KOH / NaOH if it is too acidic or too basic 6. Pour contents into glass jars, then cover with its original plastic / metal cap , and then with a piece of paper before sterilizing them in the pressure cooker at 15 psi for 15 - 20 minutes.

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 APPENDIX F PREPARATION OF WHITE’S 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

To prepare the stock solution 1. 2. 3. 4. 5.

Prepare White’s Macro Stock Prepare White’s Micro Stock Ferric sulfate stock Vitamins Stock Mix stock solutions

 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. Th e 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.

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 APPENDIX G

List of orchids which have been clonally propagated in vitro Plant

Explant

 Anacamptis pyramidalis

Shoot tip

Morel (1970)

 Aranda

Shoot tip, axillary bud

Goh (1973)

(Arachnis hookeriana x Vanda lamellate

Shoot tip

Cheah and Sagawa (1978)a

 Aranthera

Shoot tip

Cheah and Sagawa (1978)a

 Arundina bambusifolia

Shoot tip (from Young seedlings)

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

Reference

Intuwong and Sagawa (1973)a

Lindemann et al. (1970) Morel (1970) Scully (1967) Champagnat et al. (1970) Arditti et al. (1971, 1972) Ball, et al. (1971, 1972, 1973)

Cymbidium

Shoot tip

Morel (1960, 1963, 1964a,b,1970) Wimber (1963) Champagnat et all (1966, 1968) Sagawa et al. (1966) Fonnesbech (1972)

Dendrobium

Shoot tip

Sagawa et al. (1966), Sagawa and Shoji (1967), Kim et al (1970) Arditti et al. (1973), Mosich et al (1973, 1974) Singh and Sagawa (1972)

Nodal Segment Flower stalk segment (with vegetative buds) Epidendrum

Leaf tip

Churchill et al. (1970, 1972, 1973)  Arditti et al (1971,1972)

Laelia

Axillary Bud

Arditti (1977)

Laeliocattleya

Axillary Bud

Arditti (1977)

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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

Flower stalk segment (with dominant apical buds)

Fast (1973)

Phajus

Shoot tip

Arditti (1977

Phalaenopsis

Flower stalk segment

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

Weatherhead and Harberd (1980)a

Rhynchostylis gigantean

Shoot tip, lateral buds

Vajrabhaya and Vajrabhaya 1970

Schomburgkia superbiens

Lateral bud

Arditti (1977)

Vanda (Strap-Leaf)

Shoot tip Stem section

Kunisaki et al. (1972) Sagawa and Sehgal (1967)

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

DIRECTORY OF SUPPLIERS FOR ORCHID GROWING & LABORATORY ALYSONS’ CHEMICAL ENTERPRISE, INC.

Supplier of Chemical Reagents Reagents and Orchid Fertilizers 1425 G. Araneta Ave., Quezon City  Tel. 712-2266 BELMAN LABORATORIES

Supplier of Chemical Reagents, Orchid Lab Equipments and Orchid Fertilizers Belman Bldg. II, 78 Cordillera corner Quezon Avene, Quezon City Tel. 712-0201 Fax 712-0182 CHEMLINE SCIENTIFIC ENTERPRISES

Supplier of Chemicals, Fertilizer Ingredients, Lab. Equipments, Glasswares, Tools #28 Law Street, Victoria Subdivision, Tandang Sora, Quezon City Tel. Nos. 984-1198; 984-1203; 984-1201; 984-1201; Mobile 0917-3659584 Email: [email protected] DIVISORIA BOTE (ISLA BOTE) nd  Supplier of Economical 2   hand glass bottles Infront of New Divisoria Mall. Mall. Tel. 242-6244 PHILIPPINE ORCHID SOCIETY, INC. (POS) Unit 209, Delsa Mansion, No. 44 Sct. Borromeo corner Sct. Torillo Streets Brgy. South Triangle, Quezon City  Mobile: 0917-8485468 ; Website: www.philorchidsociety.org/   [email protected] ahoo.com ,  secretiat@phil  [email protected]  orchidsociety.org  Tel. 9294425; Email:  philorchidsociety@y PLANT BIOTECH LABORATORY (ORCHID LAB) RIZAL TECHNOLOGICAL UNIVERSITY

Orchid Information / Orchid Seed Culture Services Boni Avenue, Mandaluyong City Tel. 534-8267 Local 135 RAMGO INTERNATIONAL CORPORATION

Supplier of Fertilizers, Pesticides and Nursery Equipment / Tools 53 General Lim Street, Heroes Hill (Near Pantranco, Quezon Ave., ) QC Tel. 371-3463; 371-3485; 242-3370 RAMON CALADO

Orchid Seed Culture Services, Supplier of quality orchid seedlings seedlings Sumulong Highway, Antipolo City Tel. 09173364124; 457-8691

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 APPENDIX I GLOSSARY OF ORCHID TERMS  ACAULESCENT  ACAULESCENT Having no no visible stem, or a very short one  ACAULIS Having no stem  ACCRETE Grown together   ACICULAR Needlelike spine; pointed; bristle  ACINACIFORM  ACINACIFORM Scimitar-shaped. Scimitar-shaped. A "scimitar" "scimitar" is a type of curved curved sword you see in those those 1,001  Arabian Nights type type movies. I.E. curved-shaped.  ACRANTHOUS  ACRANTHOUS Term applies applies to sympodial type orchids, referring referring to the annual portions portions of  successive growth of the rhizome, each beginning with scaled-leaves, ending with an inflorescence.  ACROPETAL  ACROPETAL Leaves and flowers developing developing successively (one after the other) on one one axis so youngest is at the apex (top).  ACTIVE INGREDIEN INGREDIENT T any material in a pesticide preparation which is responsible for for the killing, suppression, or control of pests and diseases.  ACULEATE prickle-shaped  ACUMEN A tapering point  ACUTE Distinctly Distinctly and sharply pointed, but not drawn drawn out  ADNATION  ADNATION adj. Adnate Fusion of unlike parts, e.g. labellum with column; contrasted with connation  ADUMBRAL  ADUMBRAL Shady  ADVENTITIOUS  ADVENTITIOUS Applied Applied to roots which do not arise from the radicle or its subdivisions, but from from a node on the stem, etc  AERIAL ROOTS ROOTS Borne above potting potting surface  AGAR "Agar" is is just an easier way way of saying the real name "agar agar"  AGAR-AGAR  AGAR-AGAR Gelantinous substance substance obtained mostly as translucent translucent strips or or white powder from from certain sea weeds; used as solidifying agent in culture media.  ALATA Winged  ALBA Flower Flower with all segments segments white, but which may have some degree of yellow on the lip only  ALBESCENT  ALBESCENT Becoming white white or yellow  ALBINISM Lack Lack of color; deficient deficient in pigment

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 ALLIANCE Designates Designates a group of genera genera that have many common characteristics 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 differential characteristics characteristics or forms; differentiation differentiation from normal  AMABILIS Lovely  AMPHIDIPLOIDS  AMPHIDIPLOIDS – an organism organism that is diploid diploid for two genomes, each from different species.  AMPLEXICAUL  AMPLEXICAUL Clasping the stem  ANAEROBIC  ANAEROBIC Living in the absence of free oxygen  ANASTOMOSE  ANASTOMOSE When one vein unites unites with another, the connection forming a reticulation  ANGULATE More or less angular  angular   ANTHER In seed plants, part of the stamen which develops and contains pollen pollen  ANTHER CAP – A dome-shaped dome-shaped structure, protecting protecting the pollinia in in an orchid flower. flower.  ANTHESIS The period between the the opening of the flower flower bud and the withering withering of the flower or  stamens  ANTICOUS  ANTICOUS The fore-part, fore-part, i.e. that most remote or turned away from the axis  ANTRORSE Turned backwards, directed directed upwards  ANEUPLOIDY  ANEUPLOIDY a condition in which which all the cells of an organism contain an 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 MERISTEM the region of dividing dividing cells at the tip of a plant shoot. shoot.  APICULE adj. Apiculate Furnished Furnished with a short short sharp, but not stiff, stiff, point  APICULE A short and sharp, but not stiff, point  APPLANATE  APPLANATE Flattened out or horizontally expanded expanded  APPRESSED  APPRESSED Lying flat for the whole length of the organ  ARCUATE Curved like a bow  ARISTATE Tipped with bristle-like bristle-like appendage or awn  AURICLE A small lobe or ear   AUTOGAMOUS  AUTOGAMOUS Self-fertilized; Self-fertilized; flowers flowers that are fertilized fertilized by their own pollen. pollen.  AUTOPOLYPLOID  AUTOPOLYPLOID a polyploid that originates by the multiplication multiplication of one basic basic set of  chromosomes.

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 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

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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 ce ll, 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 o f  branches or in the crevices of the bark. An "a ir-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 e ither  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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MONOTYPIC One type, such as one species in a genus MOTHER PLANT – a mature orchid plants, usually in bloom, which serves as source of explant, or seeds, for MICROPROPAGATION .or conventional propagation MYCELIUM A network of hyphae made from new cells that have elongated and split repeatedly forming a network. Quiescence- A stage of dormancy that new buds enter during early winter or  periods of cold. MYRMECOPHILOUS Ant-loving; inhabited by ants NECTARIFEROUS Having nectar  NOCTURNAL Of the night; used in reference to flowers which open after dark NODE A joint or knot NON-RESUPINATE Orchid flowers normally position the lip at the bottom just above the column. Some genera, however, such as Cycnoches, Malaxis, and Nephelaphyllum position the lip uppermost with the column below making the flower appear to be up-side-down. NOMENCLATURE The system of naming ORCHIDACEAE Pertaining to a large family of perennial epiphytic or terrestrial plants; orchid family ORCHIDACEOUS Orchid family, usually having showy flowers with corolla of three petals; one labellum or lip differs greatly from others and often spurred ORCHIDIST One who collects or is interested in orchids horticulturally ORCHIDOLOGIST A botanist who specializes in the technical taxonomic study of orchids PANICLE A branching inflorescence on which all the branches bear flowers, a branched raceme. PANICULATE Having the form of a panicle. PARTHENOGENETIC Seed which develops without fertilization, but by stimulus only PEDICEL The stalk of an individual flower in an inflorescence. PEDICELLATE OVARY The combined pedicel with pedicellate of the flower  PEDUNCLE Stalk of a flower-cluster or of a solitary flower  PERFOLIATE With the leaf surrounding the stem PERIANTH Floral envelope, consisting of the calyx and corolla (even if not all parts are present) the perianth of an orchid flower consists of the sepals, petals, and lip PETAL One of the divisions or leaves of corolla PETIOLE leafstock, slender stalk by which a leaf is attached to the stem. PHALAENOPSOID Growing like a Phalaenopsis

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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 o f 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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STELIDIA Column teeth STEM PROPAGATION Small plants that are formed on flower stems. In some orchids the flower  stem has nodes which carry the dormant eyes and can develop into buds or leaves.These new plants are called "keiki".There is a hormone compound called keiki paste that is used in the developmentOf these plantlets. This practice is commonly used on Phalaenopsis. STIGMA pl. stigmas or stigmata The terminal part of the ovary, at the end of the style, which is receptive to the pollen. STIPE pl. Stipites A slender, stalk-like stem. STOMA A breathing pore in the leaf epidermis STYLE The narrow portion of the pistil which connects the ovary and the , not usually applicable to orchids. SUBSPECIES A true-breeding form of a species, often characteristic of a different geographic area, which is not sufficiently distinctTo warrant separate classification. SUBGENUS One of the divisions into which large genera are sometimes taxonomically divided. SYMBIOSIS Living together of dissimilar organisms with benefit to both. SYMPODIUM pl. sympodia adj. sympodial Orchids that produce new growth from the base of the plant where the previous growth occured (from the rhizome). The majority of orchids have sympodial growth, the others have a monopodial growth pattern. SYNSEPAL A single floral part formed by the partial or complete fusion of two or more of the orchid sepals (usually the lateral pair as in a Paphiopedilum). TAPROOT Large main root growing vertically downward. TERETE Elongate and pencil-shaped, pertaining to leaves of certain Vandaceous orchids TERRESTRIAL Plants that grow in or near ther surface of the ground; growing in soil. Orchids generally are found growing one of three ways: as TERRESTRIALS EPIPHYTES (the majority grow in this manner), or LITHOPHYTES THROAT In orchids with a tubular lip, used to designate the lower part of the tube TOMENTOSE Covered with wooly, matted hairs TRIBE A group of related genera forming a natural division within a family TUBER Thickened and short subterranean branch having numerous buds or eyes. TUBEROUS Tuber-like; furnished with tubers UMBEL An indeterminate, convex or flat-topped i` in which the pedicels of the cluster arise from a common point

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