How to make Stem Propagations

How To Make Phalaenopsis Stem Propagations

PREPARE FLASKS AND MEDIA
You have a choice of many different types of media and culture vessels (flasks) that will all work reasonably well. For flasks, I use 25 x 150 mm culture tubes (test tubes) to get things started (available from Sigma). When the plants get too big for the culture tubes they may be potted (if they are large enough and have roots) or replated to larger flasks (I use the 500 ml size).

I use both Sigma's Phytamax Orchid Multiplication Medium (product number P-6793) and Phytamax Orchid Maintenance Medium with Banana (product number P-1056). The multiplication medium is used to get things started and the maintenance medium is used for replating. I add 5 grams (approximately 1.5 tsp.) of agar (Sigma product number A-1296) per liter of medium. The multiplication medium contains plant growth stimulants which occasionally result in two to three plants per node. The maintenance medium will work fine for starting the stem props but is less likely to produce multiple plants per node.

I recommend that you prepare the media and culture vessels ahead of time. By making up the flasks a couple of weeks ahead of time, any contamination that is present will probably grow to a point that it becomes visually discernible. Flasks showing any type of contamination should not be used.

REMOVE THE FLOWER STEM
The flower stem from the plant to be propagated should be mature but not old. If the stem is very young, it will be soft and the sterilization process will kill a lot of tissue. Also, young stems tend to produce flower spikes rather than plantlets. Old stems are harder to sterilize and will be more likely to die.

Depending on who you talk to, you will get a variety of suggestions regarding when to harvest the stem. In addition, depending on genetics and other factors, the optimum time to harvest the stem will vary from plant to plant, so some experimentation will be required to obtain optimal results. As a general rule, however, I recommend that the stem be left on the plant until the first flower opens and that it be removed before the time the last flower opens.

The flower stem should be removed at its base (using sterile tools) and cut into segments with at least one node per segment. Try to leave about 1/2 inch of stem above the node and 1 1/2 inches below the node. The tip of the flower stem (or ends of any branches) may sometimes be successfully used as viable segments/nodes.

Note: All work from here requires a sterile environment (the use of rubber gloves is recommended - for your protection, not the plant's).

STERILIZE THE STEM
Perhaps the most difficult part of making stem props is to sterilize the stem without killing it. Phalaenopsis flower stems are difficult to sterilize and success can be expected about 65 to 95 percent of the time but results will vary widely. There are many types of contaminants that may find their way into the flasks, some will overtake the culture and others will not.

The following sterilization procedure is provided as a general process that yields reasonably good results:

1. Prepare three sterilizing solutions - one consisting of 15% Clorox (chlorine bleach) to 85% water and another consisting of 50% Clorox to 50% water. Add spreader sticker to each of these two solution (about 1 drop for every three ounces of solution) and mix well. The third solution consists of 3% Clorox and 97% sterile distilled water.
2. Use a toothbrush to thoroughly scrub the segments with the 50% solution. After scrubbing, immerse the segments in the 50% solution for about two minutes.
3. Rinse the segments in the 15% solution.
4. Use á sterile scalpel to remove the bract from each node (this does not apply to the terminal segments). It is particularly important to remove all of the bract tissue in order to minimize contamination rates (I use a fine pointed tweezers to accomplish this).
5. Immerse the segments in the 15% solution for fifteen minutes.

FLASK THE STEM
Remove the segments from the sterilizing solution and rinse with the 3% Clorox solution. Use a sterile scalpel to cut off the damaged (Clorox-soaked) tissue (about a quarter inch from each end).

I cut the bottom end diagonally. This produces an elliptical (elongated) surface which increases the amount of surface area that will be in contact with the medium. Next, place the segments in the medium at a slight angle with the exposed node facing up. If you are using the multiplication medium, having the node in contact with the upper surface of the medium seems to encourage the formation of multiple plantlets.

WAIT, HOPE AND PRAY
Wait six to eighteen months and pot out the stem prop if it takes; otherwise, throw it out when it turns brown.

If a flower spike begins to form rather than a plant, let the spike harden, remove the segments from the flask and cut the flower spike just above the first node that forms on it. Then put both pieces back into flasks. If the spike has more than one node on it, so much the better. Each section of the spike containing a node may also be flaked. This will significantly increase the probability of producing plants.

If you temporarily remove the segments from the medium for any reason (like replating to a larger flask), use a sterile scalpel to recut the bottom end removing any dead or damaged tissue prior to putting it back into the medium. This procedure assures that good tissue is in contact with the medium allowing the stem to absorb nutrients.

When replating, I generally wait until roots just start to emerge or until the leaves become large and very crowded in the test tube.

SOURCES
A few places that may have the supplies you will need are listed below. This list is provided for informational purposes only and should not be considered as recommendations.

For culture vessels, media and tissue culture supplies:

Sigma Chemical Co.
P.O. Box 14508
St. Louis, MO 63178
1-800-325-3010

G & B Orchid Laboratory
2426 Cherimoya Dr.
Vista, CA 92084
(619) 727-2611

ADDITIONAL READING
Over the years, The American Orchid Society Bulletin (A.O.S.B.) has published several articles dealing with flasking techniques and stem propagations. Listed below are a few of these articles that may be useful to you:

Stem Propagation of Phalaenopsis - Robert M. Scully, Jr.
(A.O.S.B. 35:40-42. January 1966) [note: this article also appears in the American Orchid Society book Meristem Tissue Culture.]

The In Vivo Propagation of Phalaenopsis Orchids - R. J. Griesbach (A.O.S.B. 53:1303-1305. December 1984)

An Easy Method to Induce Phalaenopsis 'Stem Props' - Jack Grimes (A.O.S.B. 56:369-371. April 1987)

Orchid Flasking Simplified - Vernon Stoutmyer and Ron Cooke
(A.O.S.B. 58:572-578. June 1989)

On Pollen, Pods & Props - International Phalaenopsis Alliance
(The Phalaenopsis Newsletter. Vol. 3, No. 3, pp 6,9,10)