Selasa, 08 Juni 2010

In Vitro, Orchid Cultivation


by : Peter Croezen, Green Canyon Orchids

It takes from six months to one year for an orchid seed capsule to mature. It is not uncommon for a seed capsule to contain 10,000 to 100,000 seeds. Some seed capsules may contain as many as 3,000,000 seeds. Orchid seeds are very transparent and very small, tiny specs, at times barely visible to the human eye. Seeds having the possibility for growth are said to be viable. A viable seed must contain an embryo, but the presence of an embryo in a seed is no guarantee that it is viable. Seeds may be checked for embryo presence, or absence, by placing them under a microscope.
From Seed to Community Pot

Orchid seeds have only minute reserves of nutrients and need a fungus for germination in nature, this is called "symbiotic" germination. The required nutrients are provided by the fungus. Growth of seedlings is rapid. Until 1922, it was the only known method of propagating orchids from seeds.

In 1922, Dr. Lewis Knudson of Cornell University provided us with a simpler method, "asymbiotic germination." Seeds are germinated and seedlings are grown on a sterile, jelly like, culture medium containing sugars and many other nutrients. Germination takes place without the aid of a fungus. Today, Knudson's formula "C" medium is still being used in laboratories all over the world. Since unwanted bacteria and fungi grow even faster on a sugar rich medium than orchid seedlings, all seeds, flasks, medium, and tools used must be sterilized. All the work involved, like sowing, transferring seedlings etc, is performed in a sterile environment, such as a laminar flow hood or a glovebox.

Sowing of orchid seeds in a "mother flask" is done in a sterile environment, like a laminar flow hood. Filtered through a HEPA filter, sterile air enters the cabinet and passes over the work area. Sowing of seeds is a simple procedure. Germination medium is prepared and poured into clean flasks. Flasks with medium are then autoclaved at 120º Celsius and 15 lbs. pressure for 20 minutes if small quantities of medium per flasks; longer for larger quantities. Cooled flasks are moved into the laminar flowhood where sterilized seeds are distributed evenly on top of the medium. Flasks are tightly sealed and may have a filter to allow air and moisture exchange between inside and outside. The filter must be dense enough to keep micro organisms out. The latter would spoil the germination process. Flasks are then placed under fluorescent lights for a 12 hour photoperiod, for most epiphytic species. Some orchid seeds need total darkness to germinate. After two to three months in the "mother flask", seedlings are large enough to be transferred to a richer medium and larger vessel. The transfer process is called replating.

Replating, or moving seedlings from the old vessel to another with fresh medium, is done in a sterile environment. Seedlings are replated when they have reached a stage of growth at which they will greatly benefit from being placed on nutrient richer medium. Seedlings will now grow very rapidly and usually remain in these flasks for approximately one year, or until they are large enough to be planted out to a community pot.

The community pot may hold from fifteen to 30 seedlings. Plants may spend up to one year in the community pot, before being transplanted to individual pots. After several more transplants and lots of tender loving care in years following, the orchids reach the "flowering size" stage, finally! From seed to flowering plant takes from three to seven years, depending on which orchid species seeds were sown.
Caring for Flasks

It is fun and educational watching orchids grow inside flasks. Flasked seedlings need light/dark, proper temperature and humidity. Handle flasks by grasping them near the bottom, away from the foil covered filtered top. Covered filter tops keep out micro organisms that could spoil the contents when allowed to enter the flasks.

Never place flasks in direct sunlight, unless you wish to cook the seedlings.The light should be of low intensity, 300 footcandles, gradually increased as seedlings grow bigger. Place them 30 centimeters below two four foot cool white 40 Watt fluorescent lamps that cover a growing area of two by four feet. Lights must be on 12 hours per day. Remember that 12 hours uninterrupted darkness is equally important for seedling development.

Temperatures in the 24º C (75º F) range are adequate, give or take 2º C. Keep the variation between day and night time temperatures to an absolute minimum.

Flasks that have filtered tops allow air and humidity exchange of the inside with the environment in which they are placed.To prevent the culture medium from drying out, place flasks in an environment where the relative humidity is about 60%. Keep air movement in the flasking area to a minimum. No fans!

A "flask" of small seedlings, almost ready to be compotted.
This replate of seedlings is nearly ready to be compotted. Photo by Joseph Dougherty.
Deflasking Seedlings

Seedlings stay in replate flasks for about one year, but can stay longer if the medium has not yet been depleted of necessary nutrients. Seedlings are deflasked, when leaves are at least 5 centimeters long, cleaned of all agar and transplanted to a community pot.

Pour some tepid water into the flasks and carefully swirl it to break up the agar. A spatula may be used to gently aid the breakup. Pour water and seedling mix into a bowl half filled with tepid water. Rinse seedling roots and leaves free of agar. Submerge the seedlings for about ten minutes in a weak systemic fungicide solution, like Benomyl, then place them on paper towels to dry.
Preparing Community Pots

Use a well drained, clean pot and line the bottom with drainage material. Fill the pot to within one inch from the rim with damp, loosely packed sphagnum moss. Wrap the roots of a dry seedling loosely with the same damp sphagnum moss and place the "package" in the pot. Repeat the process until the pot is filled with seedling packages touching each other. Finish by filling gaps between seedlings and pot edge with some loose sphagnum. Write the proper orchid name plus the "compot date" on plant labels and put one label in every pot.
Taking Care of Community Pot Seedlings

Seedlings deflasked have come from a 100% relative humidity environment inside the flask. Do not shock them by moving them to a 50 or 60% immediately, but rather do it gradually. This can be done by placing the pots in a tray covered with a clear plastic dome with top vents closed, which over a period of time can be gradually opened. Keep the pots about 30 cm below two 40W fluorescent lamps, as desribed above for flask lighting. Keep temperatures around the 24º C. Prevent seedling medium from drying out, but do not have it soaking wet and cause root rot. Start a regular watering program right away; fertilizing program in one or two weeks from compotting date.

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