Phalaenopsis Stem Propagation
Original Method
This is the Original Method that I had used up until about 2020. I was always trying new things to make the protocol work better with less contamination, and more plants being produced. The next section describes the Current Method that I am using.
1-Choosing Stems
It is best to use flower stems that have at least half of the flowers left in good condition. The best results are obtained when the bracts covering the nodes are fresh, and not dried and brown. To help reduce contamination, pre-bloom stems can be used once they have begun to stiffen up.
2-Cleaning stems
After removing the stem, gently clean the entire stem using a soft baby’s toothbrush, and antibacterial hand soap, wiping from the bottom of the stem towards the top of the stem. I do this under a running stream of water. Don't move the brush back and forth, as the tip ends of the bristles will puncture the stem, and may introduce contamination.
3-Removing Bract
Without rinsing off the soap residue, immerse the stem in a 10% bleach solution for 1/2 hour. During this time I use a hobby style light/magnifying glass and a pair of eyebrow tweezers to carefully remove the bract from around the node after it has been ‘ringed’ with a sharp blade. Care must be taken to remove all traces of the bract, and it doesn’t seem to matter if a portion of the “bark” of the stem has to be removed, as long as the entire bract is removed. I work over a container of the bleach solution, and often re-dip the stem and tools into the solution during removal of the bract.
4-Sterilizing Stems
The next step is to cut through the stem 30mm below the node, and 20mm above the node with a fresh single edged razor blade. The cut above the node is at right angles to the stem, the cut below the node is on a long angle to expose as much of the stem as possible which will contact the medium. The sectioned pieces of stem are placed into the plastic containers that 35mm film comes in. The containers are filled with 3% Hydrogen Peroxide, a tiny bit of the soap, and the lids are put on. The sectioned stems are left in the Hydrogen Peroxide for up to 2 hours, shaking occasionally. The container is placed into the transfer chamber, and the stems are then removed from the Hydrogen Peroxide.
5-Planting Stems
With a fresh sharp single edged razor blade trim 5mm from the top and the bottom of the stem. Without rinsing the stem, place in the jar containing the medium, with the node flush with the surface of the media. While inserting the stem into the media, push the stem deep into the gel to allow the medium to thinly coat the entire node, then pull it up flush with the medium. Cap the tube and place the stem under a single fluorescent tube for 16 hrs/day. I have found that temperatures nearing 80 degrees F are optimum for kicking the nodes into development. I am currently using Phytamax Orchid Multiplication Medium P6793 from Sigma, full strength, as per the instructions. This method reduces the time involved in rinsing the bleach solution off of the stems, and possibly introducing contamination. The bleach is neutralized by the Hydrogen Peroxide, and the Hydrogen Peroxide is neutralized by the UV of the fluorescent lights. The aggressive cleansing action of the Hydrogen Peroxide does a great job of cleaning in behind the node. Only use fresh Hydrogen Peroxide from a recently opened bottle. Air and light degrade it rapidly. Spraying the plant and stem 2-3 days prior to harvesting the stems with a systemic fungicide like Subdue 2E, makes a huge difference towards reducing systemic contamination.
Phalaenopsis Stem Propagation
Current Method
This method, 'Pat's Protocol', is the one we are currently using to do all of our stem propagation. It is the culmination of may years of research and trial and error. Most protocols yield about a 75% success rate of nodes that do not contaminate, and live to produce plants. So far on average this method yields over 90% contamination free viable nodes. This method also produces about 2-3X more viable plants per node, without mutation, than the Original Method
1-Choosing Stems
It is best to use flower stems that have at least half of the flowers left in good condition. The best results are obtained when the bracts covering the nodes are fresh, and not dried and brown. To help reduce contamination, pre-bloom stems can be used once they have begun to stiffen up.
2-Cleaning Stems
After removing the stem, gently clean the entire stem using a microfiber cloth, and Chlorhexidine antibacterial hand soap, wiping from the bottom of the stem towards the top of the stem. I do this under a running stream of water. Alternate between the soap, and the running water. Don't be too hasty to finish this step, and repeat it a few times.
3-Cutting and Sterilizing the Stems
I use glass vials with screw caps for this step. You can use whatever container you can obtain. Mine are about 90mm tall and 25mm diameter. Cut the stem about halfway between the nodes that you are going to use with a single edge razor blade. Make the cut 90* to the stem. You should now have the cut up flower stem with a piece of stem above and below each node, and the tip if it looks viable. Place the stem pieces into the vial and cover the stems with Solution #1 (they still have the bract covering the node). Cap the vial and occasionally gently rock the vials for approximately 20 minutes.
Next the exterior of the vials and everything else that is going into the glove box or laminar flow hood is sterilized with a dip into a 15% household bleach and water mixture. A microfiber cloth dipped into the bleach water and wrung out is used inside the cabinet to wipe things down in there.
These are the items that you will need inside the cabinet;
Vials of Stems, 500ml reagent bottles of Solution #1, Solution #2, Solution #3, Single edge razor blade or scalpel, flat style eyebrow tweezers, 8" long forceps, 2-500g empty yoghurt or cottage cheese type containers, and the microfiber cloth, (all processed through the bleach bath). See Figure 1
Put the stem pieces into the vial, and cover with solution #1. Gently rock the vial back and forth for 20 minutes. Dip the flask through the 15% bleach solution and place in the sterile cabinet. Next open the vial and discard the liquid into one of the yoghurt containers, leaving the stems in the vial. Rinse the stems 3 X with Solution #2, discarding the liquid into the same container. After the 3 rinses, remove the stems from the vial. Next you will use the razor blade to carefully "ring" around the bract, not cutting too deep. If you look closely at the bract, there is a delineation in color, and that is the place to cut. If you have done it correctly, you can easily remove the bract with the eyebrow tweezers exposing the node. See Figures 2, 3
Once the bracts are removed, put them back into the vial and cover them with Solution #3, and put the cap back on. Gently rock them for about 30 seconds, then remove the cap and pour out the liquid into the waste container. Rinse 3 x with Solution #2, pouring out the liquid in between rinses. Repeat one more time.
Trim the ends of the stems as shown in Figure 4. The stem only needs to end up around 20-25mm long with the node in the middle. Open the flask containing the multiplication medium and lay the stem horizontally on the surface with the node facing up. Next push the stem down horizontally into the surface of the medium until the top of the stem is level with the surface of the medium.
Using the reagent bottle of Solution #1, rinse the inside of the flask and the top of the medium including the stem. Pour out the liquid that is in the flask. Rinse the flask with Solution #2 once, and pour the liquid out. Cap the flask, and remove from sterile cabinet. Place flask under lights with the flask tilted to keep any remaining liquid away from the stem. Figure 5
Dealing with Contamination
Contamination comes in many forms, and is the #1 enemy of anyone attempting tissue culture. Yeasts, molds, bacteria, and viruses are always around us, and I am always amazed that we can actually clean a stem that has been growing in a damp greenhouse environment! Sometimes our best efforts to decontaminate the stem results in a contamination forming. If caught within a couple of days, you have about a 75% chance of re-sterilizing the stem. Because the medium using Gelrite is nearly clear, contamination is easily spotted creeping below the surface of the stem by using a magnifying visor and bright light. You can take the stem out, and repeat the sterilization protocol, and put it back into a new flask of medium. Sometimes contamination does not show up for a month, and the stems are already showing leaves. This is more difficult and you have to keep the sterilizing solutions off of the leaves, and just on the base of the node and stem.
The logic behind this Method
Although I am not an academic, I have a curious mind, and have spent most of my working career in R&D. This allows me to take a methodical and hopefully logical approach to finding the better "Mousetrap". I have a room full of antibacterial, and antifungal chemicals and have spent years trying protocols that were published in scientific papers and journals. Not a lot of luck until I started taking a bit from this one, and a bit from that one and looking at where the contamination was coming from. My original protocol is a version of how most people are doing stem props. The stems were washed, and soaked in a bleach solution and the bracts were removed so that the sterilizing solution could get to the node and clean it. Most of the contamination for me was coming from right at the base of the node. I realized that although the stem was clean, the node and stem still had a lot of contamination on it. When using the blade to remove the bract, we were actually pushing the contamination into the cut line. In the current protocol, the stem and bract are sterilized before cutting and removing the bract. This solved much of the contamination problem. At the same time we were amazed to find that using a saturated solution of Calcium Hypochlorite didn't do irreparable damage to the stem. In fact is seems to be a growth stimulator for some reason which I cannot explain, but might be related to pH as the solution is very basic. The Ethyl alcohol rapidly penetrates the cell walls of the contamination. As the Ethyl Alcohol has an affinity for water, Solution #2, (water), is rapidly drawn into the cell causing them to rupture.
Laying the stem pieces horizontally on the medium was discovered by accident as many of the novelty phals may only have one viable node very near the base of the plant. That node may only have 2-3mm of stem below the node, so it will not stand vertically as normal stems would. I used to put these nodes horizontally on the top of the medium, and they produced just as many plants. In the past the school of thought was that the basal nodes were of no value, and were often discarded. I theorized that maybe laying the stems horizontally on the medium they would be exposed to more of the growth hormones and may produce more plants, and that is what happened.
I am sure that this is not the final iteration of this protocol, so email me your thoughts and I will try to keep the protocol updated as best I can. I would be remiss if I did not mention my friends and mentors Dr. Wendy Riggs PHD, and Dr. Harry Hartmann PHD who have been there to keep me on the right path for many years!
Appendix 1
Figure 1
Figure 1
Figure 1
This image shows what you need to have in the sterile box before you start to work in a sterile environment.
- Sterile flasks with multiplication medium
- Solution #1,2,3,
- Razor blade or scalpel
- Tweezers and 8" Forceps
- Plastic waste container
- Microfiber cloth
- Stems in solution #1.
All dipped through a solution of 15% household bleach and tap water, done outside the sterile cabinet, and put inside wet.
Figure 2
Figure 1
Figure 1
This image clearly shows the light green above, and the dark green below the bract covering the node. The cut to remove the bract is made along the top of the dark green line. Sometimes the bract wraps around the back of the stem, which you can make a vertical cut parallel to the stem on the back before 'Ringing' the front of the bract. I find using 7X magnification hobby headset helps me to get an accurate cut. I hold the blade in my right hand, and the bottom of the stem in my left hand. I rotate the stem with my left, and steer the blade with my right.
Figure 3
Figure 1
Figure 3
This image shows the bract after it has been 'Ringed' with the blade, and gently pulled away from the node with the tweezers. Notice the clean cut exposing the node, and sub-node just below it. I find that the Solution #1 kind of 'pickles' the bract, and makes it easier to come off in one piece.
Figure 4
Figure 4
Figure 3
This image clearly shows the cuts on the top and bottom of the stem. I think that one of the reasons this protocol works so well is that the entire section of the stem is exposed to the hormones in the multiplication medium, producing more plantlets.
Figure 5
Figure 4
Figure 5
This image shows how I rack the flasks once the stems are put inside. The same could be achieved with a piece of plywood blocked up on one end to create the slope, or cooling the flasks on a slope so that the medium gels at a slope. What you are trying to do is keeping the remaining liquid in the flask from sitting on the stem 100% of the time. The multiplication medium is made with Gerite or Gellan gum. Although more difficult to use than Agar, it produces a nearly transparent medium which makes it much easier to deal with contamination if you can see it right away.
Figure 6
Figure 4
Figure 5
This image shows the supplies that you need to assemble for the Solutions and Medium.
-The HTH is available as a pool shock sterilizing agent. Main ingredient is Calcium Hypochlorite. HTH stands for High Test Hypochlorite. Available at most hardware and pool supply stores.
-Distilled water is available at most grocery supermarkets.
-Ethyl alcohol might be the hardest ingredient to find. It is sold through liquor stores, usually behind the counter. Some provinces or states do not carry it, but with a bit of searching you should be able to find it. It is a grain alcohol. Do not substitute Isopropyl alcohol, it will not work!
-P6793 is orchid multiplication media available from suppliers in the us and Canada.
-Young coconut. These are the ones that still have some of the husk left on the outside, and wrapped in an expanded foam sock. Watch for mold on the top as they often sit too long in the grocery store.
Solution #1
Solution #1
Solution #1
These solutions you will need to make up the day before processing the stems.
- Solution #1 is a saturated solution of Calcium Hypochlorite. You need to use a glass jar and add 40g to 500ml of distilled water. Mix well and leave the lid loosened as it will produce pressure as the chlorine is released into the water. Keep in the dark, or covered with a towel. The next day you will have a clear light yellow liquid that contains 5%active chlorine. Carefully decant the liquid from the sludge in the bottom into a 500ml autoclavable wash bottle. Leave the lid cracked a little as the solution will still gas off and push the solution out of the spout. A calcium deposit will stick to the glass jar you used to mix it up, and it is difficult to clean off. I re-use the jar for the next batch so as not to ruin too many jars.
Solution #2
Solution #1
Solution #1
These solutions you will need to make up the day before processing the stems.
-Solution #2 is sterile distilled water, (SDW). You will use twice as much SDW as the other solutions. I autoclave it in 500ml containers, and cover the top with aluminum foil once cooled to help keep it sterile. Pour into a 500ml autoclavable wash bottle.
Solution #3
Solution #1
Stem Prop Flasks
These solutions you will need to make up the day before processing the stems.
-Solution #3 is a 70% Ethyl Alcohol solution. Pour 350ml Ethyl Alcohol into a container, then add SDW to make it up to 475ml. Pour into a 500ml autoclavable wash bottle.
Stem Prop Flasks
Stem Prop Flasks
Stem Prop Flasks
Flasks are usually made a few days to a couple of weeks ahead of time. I store mine in Large Zip Lock freezer bags. This cuts down on contamination of the flasks, and also the flasks don't seem to dehydrate as fast when stored. Remember to use cling film between the Magenta B Cap and the Baby food jar.
Into a glass cooking pot I use 25g of P793 or P6793 wherever you buy it from, plus 100ml of the coconut water from a Young coconut, 3g of Gelrite, and 1l of distilled water. Bring to a vigorous boil for several minutes, stirring all the time, as the Gelrite will only dissolve at the high temperature. If some of your flasks are not gelling, it is because you did not boil it long, or strong enough.
Final Flasks
Stem Prop Flasks
Final Flasks
Once the little plantlets are starting to put roots out, they are ready to go into the final flask. I use the same medium as we use for the final replate when doing seeds. I cut them apart as best as possible, and plant them in the new flask pushing them down into the medium to get good coverage all around the base. If you get some pieces that are difficult to cut without destroying plants, I just plant those ones together and wait until next time to separate them once they are bigger. If you have difficulty getting roots to grow, make sure you are providing sufficient red spectrum which helps to initiate the root growth.