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Sample ProtocolWhen ordering media we find a baffling number of options in the catalogs. One of the most complete media is in the Sigma catalog: Murashige and Skoog shoot multiplication medium B (MSMB) (Sigma Catalog No. M7149). At the appropriate time, order a pretransplant medium (Murashige syngonium stage III Pretransplant Medium with sucrose: Sigma M 8650) You will also need a gelling agent, preferably a blend of agar and agar substitute, such as Agargel. Adjust the pH to pH 5.7 using 1N NaOH or 1N HCl (carefully, by the drop) and pH indicator paper (3.5 - 6.8), or a pH meter. (If you don't have a pH meter, the chemistry teacher might.) Now add the agar. Use about 5 grams per liter of medium . Heat and stir until the the medium is clear. (The clarity tells you that the agar has melted.) Dispense into test tubes. Sterilizing the Medium Pour about one and a half inches of water into the pressure cooker. Place the tubes upright in the cooker. To hold them upright place them in a wide mouth jar, make a wire or wooden rack, or tie them with string in bundles of ten. They must not fall over. Process at 15 pounds for 15 minutes according to the instructions which come with your cooker. Preparing Explants An explant is the part of a plant that you put in culture. The example used here is a strawberry runner tip. Select a young runner where the bud on the end has not yet opened. Cut it off the plant one inch or so from the end. Transport the tips to your classroom in a plastic bag in which you have placed a wet, but not dripping wet, paper towel. If the runner tips are not going to be processed immediately, they can be held in a refrigerator for a day or two. Fill a pint jar half full of sterile water (boiled, pressure cooked tap water or the store-bought bottled distilled water. Add 2 or 3 drops of liquid dish washing detergent (or Tween 20, a wetting agent). Place the runner tips in the jar and replace the screw-on lid. Vigorously shake the jar by hand for one minute. Pour off the water, rinse two or three times with fresh sterile water. Repeat this operation (or dip in 70% alcohol for a few seconds and rinse. In another container add 30 ml of household bleach to 270 ml of sterile water(10% bleach). To this add 2 drops of detergent. Add the explants and shake intermittently for 10 minutes. Quickly drain and add sterile water, cover and shake. The explants are now ready to take to the transfer chamber. Starting the Explants The transfer chamber should be ready with the walls and workspace wiped or sprayed with 10% bleach/sterile water solution or 70% isopropyl alcohol (not if using a Bunsen burner). There should be a container of 10% bleach/sterile water and a container of 1% bleach/sterile water to sterilize and rinse the instruments and gloved hands of the operator. Immerse the forceps and knife for 30 seconds or more in the 10% bleach then rinse in the 1% bleach and rest them on a sterile holder or paper towel to dry for a few seconds. With the forceps place a sterile paper towel on the workspace. With the forceps place a runner tip on the towel. Place the forceps in the other hand to hold the tip while the first hand uses the knife or scalpel to cut off 1 cm of the stem. Place the knife in the 1/10 bleach, move the forceps to that hand. Grab a sterile test tube of medium with the other hand, hold it by the base. With the little finger of the hand holding the forceps, remove the cap and cradle it there while you use the forceps to firmly lay the bud on the medium. Recap the test tube and seal with a piece of Scotch tape (or Parafilm). Growing Place the test tubes in the planter tray (or other appropriate holder ) and place the tray on a shelf under fluorescent light which is 8-10 inches above the top of the tubes. Room temperature is o.k. Continuous light is acceptable, but 16 hours light/ 8 hours darkness is standard. Check daily for contaminants. If any are found, sterilize tube and contents before discarding the contents. Transfer the explant every two weeks or so until it is actively growing. In one to two months you should be able to divide the culture into two pieces, each of which is about 0.5 cm in diameter. Continue to divide and transfer until you have enough plantlets. The plantlets should be singulated as you transfer to prerooting medium which has no hormones (or only IAA). Transplanting When the plantlets begin to root, perhaps two to four weeks, transplant them to a light artificial soil mix, such as peat/pearlite, in a seedling tray. Cover with clear plastic and place on a lighted shelf or in a shaded greenhouse. After two or three weeks begin leaving the plastic off for a period of time each day. The time the plantlets are left uncovered should get longer each day, until after about a week, the cover can be left off completely. (Tissue cultured plantlets are more delicate than seedlings as the stomates remain open until they slowly adjust to normal humidity and light. Conclusion After having successfully tried plant tissue culture, you will wonder about nearly every plant you see, asking yourself, "I wonder how that plant will respond in tissue culture?"
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