Standard Orchid Medium from Svante Malmgren and Henric Nystrom
(Useful for Seed Germination of Orchis, Ophrys, Anacamptis, Neotinea, Gymnadenia)
The following media can be used for seed germination of the above orchid genera. This medium is slightly modified from Malmgren and Nystrom to reflect the fact these authors use a solution of amino acids called "vaminolac". Vaminolac is not available in the United States. As a substitute for vaminolac they suggest using inorganic ammonium nitrate and ammonium phosphate to provide the protocorms with a source of nitrogen. They note that some species do better with vaminolac and some better with inorganic nitrogen. In this modification to their medium I have chosen to compromise by using casein hydrolyzate as a source of amino acids plus adding ammonium nitrate and ammonium phosphate at about 1/2 their original concentrations. Thanks are extended to Svante Malmgren and Henric Nystrom for making the formulation of their media available to all.
1. Ingredient Amount per Liter
Ca5OH(PO4)3 75 mg (original recipe calls for Ca3(PO4)2 )
KH2PO4 75 mg
MgSO4x7H2O....................................75 mg
Sucrose 12 grams
Activated Charcoal (fine) 750 mg
Agar 5 grams (Agar, plant Phytotechnology Labs #A-111
Casein, Hydrolyzate 200 mg (N-Z-Amine A Sigma Aldrich #C7290)
NH4H2PO4 100 mg
NH4NO3 50 mg
Pineapple juice 20 ml (fresh squeezed or frozen fresh squeezed. Canned juice or
............................................................dried pineapple powder is available but suitability is
...........................................................unknown.)
Add water to 1000 ml total volume in a volumetric flask.
2. After dissolving all ingredients at room temperature adjust pH with a pH meter to pH 5.7 to 5.9. The initial pH may be quite close to pH 5.7-5.9. If not, adjust with HCl or KOH as appropriate.
3. Heat all ingredients to boiling in a pyrex beaker to dissolve the agar which will not be dissolved prior to this time. Aliquot the boiled and dissolved ingredients into desired culture vessels in preparation for autoclaving. Into each aliquot of media add 1 cm3 russet potato cube per 20 ml of media. In my situation, I plate my seed onto 150 mm diameter Falcon petri dishes which hold approximately 100 ml of media. Thus, I use 5 cubes of russet potato. Because the Falcon petri dishes are not autoclavable I autoclave the media + potato cubes in pint wide-mouth Mason (canning) jars. Autoclave for 30 minutes at 15 psi. Pour autoclaved media into the petri dishes or other culture vessels. Leave lids off petri dishes or culture jars until media has gelled and cooled. This will avoid water condensing on the lids which makes observing bacterial/fungal contamination difficult. If using petri dishes, seal edges with parafilm (Phytotechnology Labs # S801).
4. With the modification of the original Malmgren and Nystrom media the following species have been germinated: Anacamptis picta, Anacamptis longicornu, Anacamptis champagneuxii, Anacamptis sancta, Anacamptis laxiflora, Anacamptis morio, Anacamptis palustris var rosea, Anacamptis syriaca, Orchis punctulata, Orchis provincialis, Orchis italica, Orchis quadripunctata, Dactylorhiza incarnata, Dactylorhiza purpurella, Dactylorhiza saccifera, Dactylorhiza sambucina, Platanthera bifolia (Scientific terminology follows Bateman et al., 2003)