Exercise B Clones From Tissue Culture

  1. It is important to stress sterile technique, since the cultures can casilv become contaminated. You should m demonstrate how to sterilize instruments by dipping in alcohol and passing through a flame. Explain the safety precautions ncccssary when using alcohol near an open flame. If a laminar flow hood is available, have the students use it. This will dramatically cut down on the contamination. If not, make sure the students clear their lab benches and wipe the counters down with 70% ethanol. If it is possible to work in an area with limited air flow, this will also cut down on contamination. Nevertheless, many students will have contaminated cultures. One reason for using 3 or 4 pctri dishes is to increase the chances of having one uncontaminatcd plate. Note: If you arc using a laminar flow hood, make certain you use a shielded burner not a regular bunscn burner.
  2. Tobacco works very well for tissue culturins, and tiiis

is a good opportunity to explain the value of tobacco as an experimental tool. Although students hear a lot about the negative aspects of tobacco, rhcy should realize its positive value in research. The hormone concentrations given in step 4 are diosc specifically for tobacco callus. If you use other plants, you may need to find the correct concentrations for that plant. When cutting sections of tobacco stem, make sure the students avoid the nodes. Culturing works best with parcnchyma cells in the pith. When the students trim off the epidermis, they can also remove the cortex and vascular tissue as well, leaving just the thin cylinder of pith.

  1. For culture media, the same basal medium is used for die callus growth medium and the differentiation medium; only the levels of hormones differ. The callus growth medium is poured into petri dishes. You can use test tubes, but it is more difficult for students to retrieve the callus from the tubes. For differentiation of shoots and roots, the medium should be dispensed into magenta jars. If these are not available, you can use baby food jars. Both need to be sterilized before you pour rhc media.
  2. Media can be made from scratch, but it is more convenient to use prepackaged media. Murashige and Skoog basal medium with sucrose and agar is available from Sigma (M-9274). Each package makes one liter of medium, which is enough for 50 petri dishes or about 20 magenta jars. Follow the directions on the package to prepare the medium. Adjust the pH to 5.8 with IN XaOH prior to autoclaving. Place a stir bar in the tlask before autoclaving. After autoclaving, add the hormones as follows: Both hormones should be separately dissolved in 1 or 2 ml of IN NaOH (they arc not water soluble) and filtcr-stcrilized prior to adding to the culture medium. Allow the media to cool somewhat (but don't let it solidify). Place the culture medium tlask on a magnetic stir plate and stir at medium speed for about one minute to distribute the hormones. Use culture media within a week of preparation. Prolonged storage can cause the hormones to degrade.
  3. For die callus growth medium, use 2.0 mg IAA per liter of medium and 0.2 mg kincrin per liter of medium.
  4. For the differentiation medium, use 0.02 mg IAA per liter of medium and 2.0 mg kincrin per liter of medium.
  5. Ideally, cultures should be incubated in a growth chamber, but excellent results can be achieved using a bank of cool white fluorescent bulbs at room temperature in the lab.

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    How to prepare murashige and skoog media from scratch?
    8 years ago

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