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.

Macronutrients                                                   Amount

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.

****Important Note: The "R" medium is often used as a "Replating" medium in addition to a "Plating" medium. For replating reduce the number of 1cm potato cubes to 1 cm cube per 200 ml of replate media. This lower concentration is better for root growth.

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