Propagating Orchids from Green Pods
We have processed many "green pods," actually immature capsules, from Cypripedium and fewer from other species. The key to success with green pod propagation is to process the pod when the embryos have matured to the point at which they can carry on development in flasks but to not wait so long that the embryos have matured into seed which require more complex treatments, including vernalization, to break dormancy.
If successful, the use of green pod propagation may save a year in achieving a blooming plant compared to the use of mature seed. This is accomplished by avoiding the added time for seed to become fully mature and several months of vernalization. We always attempt at least one green pod culture for any new cross that we attempt since many crosses result in viable embryos that subsequently die during the maturation process. It is found with many orchids and other plants that "embryo rescue" from green pods results in the embryos successfully growing on to adulthood. Of course, progeny of some crosses will not survive to adulthood due to genomes that are too incompatible.
Timing of Green Pod Collection
Timing of the collection of green pods is an empirical process and many tables exist with estimates. Fortunately for those interested in Cypripedium, maturation of the pods is extremely rapid. Green pods are optimally collected between weeks 5 and 8 following application of pollen. The exact time is affected by the weather during maturation, with warm temperatures speeding maturation and cool temperatures delaying maturation. We have found empirically that if we harvest green pods 6 weeks following pollenation that we always have reasonable survival of the embryos. It should be kept in mind that this timing is appropriate for northwestern Connecticut and will be somewhat shorter in warmer areas and somewhat longer in cooler areas.
Processing Green Pods
We have used a modified Harvais medium for green pod culture of Cypripedium. Have 4 flasks of medium on hand for each pod to be processed. For some species that produce large pods, e.g. Cypripedium reginae, 6 flasks can easily be filled.
Outside of the sterile hood or box:
| 1. |
Examine the pod carefully for insect holes or fungus or bacterial holes that may extend through the pod wall. If these exist the pod should be discarded as the embryos will be contaminated and sterilizing fluid will enter the pod and kill the embryos. |
| 2. |
Prepare 200 ml of Chlorox bleach:water in the ratio 100 ml Chlorox:100 ml water. |
| 3. |
Add to the solution 4 drops of Tween 80 or Ivory dishwashing liquid. Stir. |
| 4. |
Remove any dried petals and long stem fragment using a scalpel or razor blade, leaving only stumps. Do not cut too close to the pod or sterilization fluid may enter, killing the embryos. |
| 5. |
Place the green pod into the bleach:water solution and scrub lightly with a toothbrush. |
| 6. |
Incubate the green pod in the bleach solution for 15 minutes with occasional stirring. |
Inside of the sterile hood or box:
| 7. |
Inside the hood place 3 sterile containers, 2 containing 100 ml sterile water and 1 containing 100 ml 91% isopropyl alcohol. Also have a square of sterile glass, 4 to 6 inches (10-15 cm) on a side. |
| 8. |
Using forceps, remove the now-sterile pod from the bleach:water solution and place it into the first container of water. Swirl the pod gently for 2 minutes to rinse off the bleach. |
| 9. |
Using sterile forceps, transfer the pod to the second container of water and swirl for 2 minutes. |
| 10. |
Transfer the pod to the container containing alcohol and swirl for 2 minutes. |
| 11. |
Place the pod onto the sterile glass to allow the alcohol to evaporate (some workers burn off the alcohol at this time; we do not). |
| 12. |
Using a sterile blade, remove both ends of the pod. Dip the blade into alcohol and allow to dry, then slice the pod lengthwise into 2 halves. |
| 13. |
Using sterile forceps hold a half-pod over an open flask. Use a sterile needle to scrape about half the embryos into the flask. If the cut surface of the pod is glistening white rather than filled with fluffy embryos, use the needle to scrape about half of the placenta containing the immature embryos into a flask. |
| 14. |
If the surface of the media is "dry" add about 0.5 ml sterile water, close the flask, and swirl it gently to disperse the seed. |
| 15. |
Seal the flask with Parafilm or plastic stretch sealer. Label the flask lid using a permanent marker. Include a description of the contents and the date this mother flask was made. |
| 16. |
Place the mother flasks into a dark cabinet at room temperature. If possible this should be 70 - 75 F to produce maximum growth rate. |
