William Steele "R" Medium
The William Steele medium is an excellent medium for germinating the seed of most Cypripedium species and also many other terrestrial orchids. Of the many ingredients most are inorganic ions. The ingredients are divided into "macronutrients" and "micronutrients". Because it is difficult to weigh out the small quantities of micronutrients they are made up in a 1000X stock solution. For each liter of media use 1 ml of the 1000X micronutrient stock solution. The ingredients listed below are for 1 liter of medium.
1. Casein Hydrolyzate 500 mg
2. Calcium Nitrate, Ca(NO3)24H2O 400 mg
3. Potassium Phosphate, KH2PO4 200 mg
4. Magnesium Sulfate, MgSO47H2O 200 mg
5. Potassium Nitrate, KNO3 200 mg
6. Potassium chloride, KCl 100 mg
7. Ammonium Citrate, C6H14N2O7 19 mg
8. Ferric Ammonium Citrate 25 mg
9. Myo-inositol 100 mg
10. Glucose 20 grams
11. Agar 7 grams
12. 1cm3 Russet potato cubes 40
13. 1000X micronutrient stock solution 1 ml
14. Reverse Osmosis or Distilled water make up to 1 liter in volumetric flask
Micronutrient Stock Solution 1000X***
Ingredient Amount in 1 liter of Stock Solution
1. Boric Acid, H3BO3 500 mg
2. Copper Sulfate, CuSO45H2O 25 mg
3. Zinc Sulfate, ZnSO47H2O 500 mg
4. Sodium Molybdate, NaMoO42H2O 20 mg
5. Cobalt Nitrate, Co(NO3)26H2O 25 mg
6. Potassium Iodide, KI 100 mg
7. Manganese Sulfate, MnSO4H2O 1540 mg (1.54 grams)
8. Reverse Osmosis or distilled water make up to 1 liter in volumetric flask
Mix all 14 macronutrients (except Russet potatoes) in a 1 liter volumetric flask. Dissolve all ingredients that will dissolve by stirring (the agar won't dissolve). Pour into a large beaker and adjust to pH 5.7-5.9 (if necessary) with 10 N NaOH using a pH meter. The pH may be within limits without any adjustment. Heat mixture until boiling to dissolve the agar. Pour heated mixture into suitable autoclavable containers. For each 25 ml of media add 1, 1cm3 Russet potato cube(this is equivalent to 40 cubes per liter as above) to the containers. Autoclave media for 30 minutes at 15 psi in a pressure canner. Remove from pressure canner and cool. Normally, I autoclave 100 ml of medium in pint canning jars. I pour this cooling media (but not cold media) into 150 mm petri dishes which can easily hold 100 ml of medium. Leave lids off until the medium has cooled and solidified. This will prevent condensation on the lids of the petri dishes.
Some protocols use various cytokinin hormones. I often use the hormone meta-Topolin at a 2 uM concentration. This horomone is heat labile and should not be autoclaved. So, I add the meta-topolin after autoclaving but before the media has solidified. You can purchase filter sterilized meta-topolin from Phytotechnology Labs in a concentration of 1mg/ml. For 100 ml of media add 50 ul (microliters) of the 1mg/ml solution to produce a 2 uM concentration of the hormone.
***Store the 1000X micronutrient stock solution in the frig. It will last for several years at least.
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