Make water/agar media by adding 10 grams of agar to 1 liter of water (note: no growth salts or sugar is used). Fill pint jars with 30 ml of water/agar media. Autoclave and allow to partially cool under a laminar flow hood. Before the water/agar gels (~ 40C), add 3 ml of colchicine stock solution cold filtered through a .2u nylon syringe filter, into each jar. Final colchicine concentration will be ~.05% w/v. Cap with a solid lid. Cover jars with aluminum foil to protect from light.
Choose protocorms for conversion at a development stage when they have chloroplasts (i.e. greened up). For Odonts, diameters are typically 2-3 mm with the signs of a leaf primodia just beginning to develop.
Treat protocorms by placing them on this colchicine substrate for 3-5 days. After treatment, remove and spread onto replate media. A simple hook is all that is needed to remove the protocorms from the colchicine media which has been made stiff on purpose. I do not rinse the protocorms off. I "plow" protocorms into the surface of the replate media to make sure protocorms are in close contact with the media.
There is a long recovery period for treated protocorms before they resume growing. Some or many will die (sometimes all them die). After growing on I select plants for final replate. When replating, I search for plants with the bluntest leaves (rounded ends versus pointed ends) for final replates.
Polyploid Odonts will typically grow as fast or faster than diploid plants. I mention this fast growth because it came as a surprise to me. Precedent growers told me to always save the slower growing seedlings because, "they're the tetraploids" - nonsense, they'r the junk!.
Conversion rates are around 25%.
Bob Hamilton - May 95