This is the medium normally used at Sapphire Dragon Orchids for doing Phal stem props. I generally get 5 or more plantlets per node using this method and I have had no problems with mutation.
Base Ingredients for 1 Liter Final Medium:
- 20g/L P793 - PhytoTech Orchid Multiplication Medium without Charcoal and Agar
- 100 ml/L Coconut Water
- 10 mg/L BAP (10ml stock solution, see below)
- 4g/L A133 - Agargellan
- 890ml Distilled Water
Add the ingredients to the distilled water and bring to a rolling boil to make sure the Agargellan has dissolved fully. You can then pour the liquid medium into stem prop tubes or other autoclaveable containers. Then autoclave as you normally would any other flasking medium to sterlize them.
This brings the BAP level up to 12mg/L, P793 just does not have enough on it's own. You can play around with the amount of BAP. 10mg/L is generally the amount most people use. I've had no problems pushing it up to 12mg/L and I'm usually getting more plants per node. You can also use O139 if you want more control over the BAP. But you may have to add some NA back in.
No matter what basal medium you use, you will have to play with the amount. The recommended amount of 25g/L of the P793 is too hot for me. So I tend to drop down to 20g/L.
I have had very good luck working using Agargellan from Phytotech as my gelling agent. I like it more than the other gelling agents I've tried.
I use a large volume of Coconut water in my medium. The amount of Distilled water above is adjusted for the amount of other ingredients to get you very close to 1000ml of final medium. If you change the amount of BAP and/or Coconut, be sure to adjust the amount of water you add to bring the final volume up to 1000ml.
Directions for making BAP Stock Solution (1mg/ml)
- 100mg BAP (Benzylaminopurine)
- 1ml 30% KoH (Potassium Hydroxide)
- 99ml Distilled Water
BAP will not dissolve in water, so KoH is usually used as the solvent. Add the 100mg of BAP to 1ml of KoH. Carefully stir the mixture until the BAP has fully dissolved. The slowly add the 99ml of distilled water.
At this point I usually place 10ml of the stock solution into small vials and freeze any of them that I am not going to immediately use. Then when you need to make a batch of stem prop medium, all you need to do is thaw one vial of the stock BAP solution.