Harvais medium from stock solution

Originally cited by Harvais, 1974. Notes on the biology of some  native orchids of Thunder Bay, their endophytes and symbionts.  Canadian Journal of Botany, 52, 451-60.

Amount in Media Concentration of Stock Solution Volume of Stock per Liter of Media  Ca(NO3)2 * 4 H2O400 mg  8.0 g/200 ml  10 ml  NH4NO31400 mg  28.0 g/200 ml  10 ml  KH2PO4200 mg  4 g/200 ml  10 ml  MgSO4 * 7 H2O200 mg  4.0 g/200 ml  10 ml  KNO3200 mg  4.0 g/200 ml  10 ml  KCl 100 mg  2.0 g/200 ml  10 ml  Ferric ammonium citrate 25.0 mg  Ammonium citrate 19.0 mg  0.50 g/200 ml  0.38 mg/200 ml  10 ml (both in same stock solution)

Microelements + Mn (All in same stock solution)

H3BO30.5 mg  500 mg/L  1 ml  CuSO4 * 5 H2O0.025 mg  25 mg/L     ZnSO4 * 7 H2O0.5 mg  500 mg/L     Na2MoO4 * 2 H2O0.02 mg  20 mg/L     Co(NO3)2 * 6 H2O0.025 mg  25 mg/L     KI 0.1 mg  100 mg/L     MnSO4 * 4 H2O1.54 mg  1540 mg/L

Glucose  20 g (Steele)  Agar  7 g (Steele)

Kinetin may be added, if desired, at 0.5 to 1.0 mg/l, and should be  filter-sterilized (according to Harvais). Steele notes that more may be added, and it may be added to solution before autoclaving. Kinetin  may be dissolved by adding 25 mg kinetin to 1.5 ml of 0.1 normal HCl, heating and agitating, and then diluting to 100 ml with distilled water. 1.2 ml of this solution to 250 ml of mineral medium provides 1.2 mg/l of kinetin.

Adjust the pH to between 5.7 and 6.0 with 0.1 normal NaOH, or 0.1  normal HCl.

Potato cubes (1-2 cubes 5mm on an edge per 25 mm culture tube) may be  added to optimize culture of Cyp. reginae. Cubes are added to the  tube, the media is poured over them, and the container is capped and autoclaved.

Note that while Harvais (1974) recommends 800 mg/L of calcium  nitrate, his 1982 formula uses the 400 mg/L as noted above.

Source: Steele, W. K. 1995. Growing Cypripedium reginae from seed. American Orchid Society Bulletin 64: 382-391.