Dr. Toby's Laboratory Learning Techniques and Tips

Preparing Plant Tissue Culture Medium

In the plant tissue culture process, plant parts are cut into small pieces that will give rise to many more individual plants. Soil or hydroponically grown plants only need fertilizer (a source of K, N and P plus trace minerals), water, air and light to grow, because they can make their own sugar, amino acids, etc.

Plant tissue cultures need an outside source of sucrose because the tissue culture cells go into shock when the explants are made. It helps to add vitamins in addition to the fertilizer and sucrose.

We focus on Stage I (cell-multiplication) medium for the introductory tissue culture process. Stage II medium varies.

Pre-mixed plant tissue culture media are available from:

• Carolina Biological Supply Company (violet/ gloxinia multiplication medium)(you may need to login)
• Sigma Chemicals MS basal salts, MSMA (you may need to login)
• Life Technologies/Invitrogen MS basal salts or plus vitamins (you may need to login)
• Kitchen Culture Kits

Other plant tissue culture media include Gamborg's. For African violets, Basal Murashige-Skoog salts (MS) suffice. To see formulations,(i.e. the complete composition of the tissue culture medium, click here or here. Here's a cool method for the classroom. You can turn this method into an experiment on formulating tissue culture medium from household materials (fertilizer, people vitamins, sugar, pudding agar-agar. Folks involved in kitchen tissue culture often use this approach.

Pre-mixed media can be purchased in packs for preparing 1 liter. One liter usually provides enough medium to fill 60 50-ml tubes with 15 ml. Some folks like to use deep Petri plates (20X100mm). These need about 25 ml of medium per plate.

Is sugar in the pre-mix? Generally the pre-mixed formulations DO NOT contain sugar, nor do they contain agar. A simple way to figure out if the formulation packet contains sugar is that the total weight on a 1 liter packet would be around 30 grams. If sugar is NOT included, the total weight will be less than 5 grams. Generally, folks add 20-30 grams of sucrose to plant tissue culture medium.

What agar to use? Agar is left out because these pre-mixes are often used for suspension cultures. Note that Nutrient Agar contains lots of food for bacteria in addition to the inert agar. Be sure to use AGAR (not nutrient agar). Gel-Rite and Phytagar are more pure preparations used by professionals. Gel-Rite is used at lower concentrations (grams per liter or %) than agar. Bacteriological agar works fine for schools. Typically, agar is added at 0.7%-0.8% or 7-8 grams/liter.

Preparing the Plant Tissue Culture Medium

No autoclave? Your local hospital might accommodate you. Plant tissue culture medium MUST be sterilized since the cultures will be in progress for much more than 8 weeks. Contrast bacteriological medium which CAN be boiled to sterilize and used within a couple of days because the microbe cultures grow up overnight.

Materials

Procedure for 1 liter (40-60 samples)

1. Add about 800 ml of distilled water to the 2 liter flask. You need a 2 liter flask for 1 liter of medium to contain boil-overs that will occur during the sterilization process.
2. Add the contents of the packet. Use a dropper to squirt some water into the packet to flush out all of the powder. Swirl to mix completely or put on the stirrer. When ALL of the powder is in solution...
3. Add sucrose and swirl or stir to dissolve the sucrose completely.
4. Check the pH (NO do NOT add the agar until after you adjust the pH). The pH will be around 5-5.5. Though plants generally like acid soil, this pH is too low for the agar to gel.
5. Adjust the pH to 5.7. NOTE: the plant tissue culture medium is NOT buffered so add the base or acid in small portions (about 1 ml per dose).
6. Add distilled water to the 1 liter line on the Erlenmeyer flask.
7. Add the agar (7-8 g/liter). The agar will NOT dissolve.
8. Cover with 2 layers of aluminum foil and put a piece of autoclave tape on the label area of the flask. Autoclave for 15 minutes at 15 psi ( standard autoclave conditions). If available, use slow exhaust.
9. If you are using glass tubes with Magenta-brand or Kimax brand plastic closures, rinse the tubes out with distilled water (OK to have a tiny bit of water residue in the tubes) and autoclave with the culture medium (with caps ON of course). If you are using disposable 50 ml centrifuge tubes or plastic Petri plates, do NOT autoclave these for they will melt and smell terrible aside from really mucking up the autoclave.
10. Cool to about 60C.
11. Aseptically, pipette or pour the warm liquid medium into the sterile plastic or sterilized glass culture vessels in a hood. The gel will set in about 1 hour. See a simple hood design for aseptic work.
12. Store, refrigerated after the medium has completely cooled. Wrap the entire rack in plastic (e.g. use a large plastic baggie) to keep the medium from drying out.

Turn this into a lesson by having students look up the formulations on the Invitrogen site, then figure out the molarity of each component.

Ask students to bring in fertilizer, read the label, and then compare the MS formulation with the composition of the fertilizer. They can do the same with vitamins in a pill!

Unfortunately, as with many culture media recipes, some components are listed in %, some in molarity and some in ppm! This generates a real "need to know" the math and chemistry and unit conversions for students, for we scientists have to be flexible and know how to work the numbers, ourselves!

Remember, a 1% solution (w/v) is 1 gram in a total of 100 ml or 10 grams in a total of 1 liter. Molarity is weight of the component divided by its molecular weight. ppm is....

Adding Hormones

Auxins, kinetins and gibberellins are the main types of plant hormones; one stimulates roots, another shoots and gibberellins stimulate internode growth. Generally these hormones can be added BEFORE the medium is sterilized. Usually we make a stock solution which we store frozen. Generally 1 mg/ml or 10 mg/ml stocks work fine, since most of the hormones are needed in very low concentrations, like 1 mg per liter. Making up the stock solution varies for each hormone. Some have to be dissolved in a very concentrated way using acid or base, then brought to volume with distilled water.

I generally weigh out between 10 and 20 mg and get the exact amount---say 13.6 mg. I actually weigh the hormone right into the tube where I will prepare the stock. For a 1 mg/ml stock, where I have weighed out 10 mg or more, I would add about 1 ml of distilled water, then adjust the solubility by adding small amounts of 1 M acid or base until the powder actually dissolves, then increase the volume to bring it to the needed total, in this example, 13.6 ml. 15 ml plastic centrifuge tubes are great for this! Then, I would aliquot the material in 1 ml portions in microcentrifuge tubes to store frozen. When preparing the next batch of plant tissue culture medium, thaw and add 1 ml for each liter, or try making smaller volumes to add to each individual culture tube for a dose-response study.

For example, if the hormone dose called for is 1 mg/ liter and I have a tube with 15 ml of medium, I would add 15/1000 of 1 ml or 15/1000 of 1000 ul or 15 ul (remember to use sterile pipette tips). You can safely add up to 0.2 ml (200 ul) per 15 ml without greatly affecting the concentrations of other components. I would add the hormone the day before I planned to use the tube so that the hormone has a chance to diffuse throughout the culture medium. You can also prepare intermediate dilutions (keeping everything sterile) so that whatever dose you add is delivered in 0.1 ml portions. Students should try powers of 2-5, both below and above the recommended dose. They may find, much to their surprise, that higher doses are toxic or inhibitory, giving credence to the notion that too much of a good thing can be bad! For this, I'd prepare some 10 mg/ml stocks.

References

1. Plant Tissue Culture Information Exchange
2. Suppliers
3. Plants From Test Tubes (Kyte and Kleyn)