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Protocol For Colchicine Treating Protocorms

1) Make a stock solution of .5% colchicine weight by volume by adding 1 gram of colchicine to 200 ml. of distilled water. This stock solution should be cold filtered or autoclaved and stored in an amber bottle protected from light. I keep my solution refrigerated. Colchicine stock solutions are subject to contamination by fungi. Note: colchicine is ranked highly toxic. 2) 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. 3) 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. 4) 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. Conversion rates are around 25%. Bob Hamilton - May 95

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