Making Tissue Culture Media

1
Making Tissue Culture Media

• Dayton High School
• Tissue Culture Lab

2
Making Plant Tissue Culture Media

• Lesson 2
• Goal The student will be able to recognize the
  ingredients needed to produce a proper plant
  growing media.
• The student will be able to explain what Plant
  Growth Regulators are and what effects they have
  on the plants in tissue culture.

3
Clean Water

• A deionized/filtered water supply may be needed.
• Chlorine and Floride at low levels is ok, but at
  higher levels is toxic.

4
Plant Nutrients

• Nutrients needed are supplied by many of the
  media recipes available.
• pH range of the media is usually between 5.0-6.0

5
Solidifying Agents

• Agar, phyta gel, gelrite etc.
• The matrix formed to support plant but allow for
  diffusion of nutrients of the plant.
• Other alternatives include glass wool, filter
  paper, silica gel, starch/pectin.

6
Possible Deficiencies

• Incorrect media may lead to deficiencies.
• Chlorosis- when nutrients are being depleted. See
  Picture.
• Shoot Tip Necrosis- Commonly a calcium deficiency
  in new growth.
• Calcium also carries other nutrients.

7
Carbohydrates

• There is little photosynthesis during tissue
  culture. Carbohydrates must be provided in the
  media.
• Sugars used in the media include glucose,
  sucrose, fructose, sorbitol, etc.
• Some plants grow better with one type of sugar
  than the other.

8
Vitamins Needed

• Vitamins are important for plant growth and
  metabolism.
• Commonly includes Thiamine, Nicotinic Acid,
  Pyridoxine, and Inositol.

9
Plant Growth Regulators (PGR)

• Cytokinins- 6-Benzylaminopurine (BAP), Kinetin,
  Thidiazuron (TDZ), Zeatin-
• Stimulates cell division and shoot multiplication

10
More PGRs

• Auxins- 2,4 Dichlorophenoxyacetic acid (2,4-D)
• Indole 3 Acetic Acid (IAA),
• Indole 3 Butric Acid (IBA)
• Napthaleneacetic acid (NAA)

• High levels of these PGRs promote rooting, and
  lower levels promote shoot elongation, apical
  dominance, and callus initiation.
• Gibberellic Acid (GA) promotes cell elongation

11
Stock Solutions

• It is possible to make a generalized stock
  solution, but not all nutrients can be put into
  the solution without some nutrients precipitating
  out.
• It may be necessary to make a small stock
  solution for specific plant types, or purchase
  pre-made solutions.

12
Review Questions

• 1. What type of water supply may be needed to
  make plant media?
• 2. What is the typical range in pH for plant
  media?
• 3. List two types of solidifying agents. What is
  their general purpose?
• 4. What can lead to nutrient deficiencies?

13
Review Continued

• 5. Explain why carbohydrates need to provided in
  the media. What sugars are typically used in the
  media?
• 6. Why are vitamins important to plants?
• 7. What does PGR stand for? What is its purpose
  in plant tissue culture?
• 8. Is there a perfect stock solution for all
  plants? Explain why or why not.