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Hi Guus and others who do sterile in-vitro work. I know that this thread has been inactive for years but it has been viewed many times so I thought that it may still be OK to post a reply. I used to use hypochlorite as it is such an efficient desinfectant, but it is also quite a nasty compound from a toxicological point of view. I have therefore made a cheap and simple sterile cabinet for handling my orchid seedlings, so now I only use very small amounts of hypochlorite for sterilising the seeds before plating them. My sterile cabinet was very easy to build, I have given the details here: http://invitroorchids.dk/en_index.htmlAnders
Hi Corrado and RinaI have tried to give as many details as possible on my homepage so I will only give some short answers here. I am an environmental microbiologist, so the way I grow orchids from seeds is basically the same as growing bacteria in the lab. There is no door in front of the cabinet, only a polycarbonate glass plate (plexiglass) and an 15 cm open slit at the bottom, 15 cm is enough to handle things inside the cabinet and to get things in and out. The flow of sterile air from the air purifier at the top is enough to keep the kabinet sterile. As containers, I use glass jam jars from the kitchen. No holes and no ptfe filters. The jars are sealed with two layers of clingfilm which is permeable to oxygen and ethylene, but not to water molecules. I do sterilize the jars with the medium in them (each covered with tin foil) by cooking them 30 min in a pressure cooker. But that is only when I make few jars, most of the time I sterilize the tinfoil covered jars for 60 min in a 200 degree C oven, sterilize the medium in 0.5-L bottles covered with tin foil - no closed bottles in the pressure cooker!!! - and pour the medium into the jars in the sterile cabinet by gently lifting the foil a few centimeters. I use the same medium for all orchids, my recipe is simple (see my homepage) and based on liquid fertilizer for indoor plants. Some orchids probably grow better on Malmgrens medium with inorganic nitrogen, I guess you already know Malmgrens homepage where you can find his recipe. I haven't had any success with European Epipactis except palustris from very fresh seeds. For the Cyps, I always cross-pollinate two genetically different clones, selfing gives way bad germination and growth for most cyps. AndersPS MAlmgren homepage http://www.lidaforsgarden.com/Orchids/engelsk.htm
The addition of fruit juice (mostly orange or pineapple) serves at least four purposes. The most important is to add buffer capacity so that the pH remains constant during seedling growth. Plant cells excrete H+ ions (acid) when they take up nutrients, this means that the medium will soon be very acidic and toxic if there is no pH buffer. Fosfate has some buffer capacity but is relatively small at pH 5.8. Fruit juices contain citric acid and other organic acids that are very efficient buffers at pH 5.8. It is of course very important to adjust the pH of the medium to around 5.8 AFTER adding the fruit juice. The second purpose is to keep micronutrients, especially iron, in solution so that they are available to the plant cells. Citric acid and other organic acids form soluble complexes with iron and other micronutrients. Without the citric acid, iron will precipitate as insoluble iron hydroxides (rust) that the plants cannot take up. The third purpose is that fruit juices contain a complex mixture of growth factors (hormones, vitamins, trace elements etc), but the extra plant hormones are not always beneficial, some species can be very sensitive to the wrong combination of hormones. And least important, both orange juice and pineapple juice contain enzymes (proteases) that hydrolyzes proteins into free amino acids and peptides that can be taken up by the plant as an organic nitrogen source. This effect counts only when there is a protein source the medium, for instance in the commercial modified Malmgrens medium where free amino acids are substituted with peptone (partly hydrolyzed protein) or if blended vegetables are added. The enzymes are only active before the medium is heated in the pressure cooker. Canned orange juice and canned pineapple juice havealso been heated which inactivates the enzymes. Pheew, this became a little too technical.Anders
The agar content should be as low as possible, but the gel should also be strong enough to support the seedlings. The brand I use (from a Chinese food store) is good, 5 g/L is sufficient. For other brands, it may be necessary to add 7 or 8 g/L.
