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CELL TRANSFORMATION IN TOBACCO LEAF DISKS

Trish Russell
1993 Woodrow Wilson Biology Institute


INTRODUCTION

This is a relatively simple plant transformation experiment. Students are working with whole plant material and are not required to measure small quantities, yet they can see evidence of transformed plant cells (plant cells that have genes from bacterial plasmids). The experiment can be extended for further work with the transformed cells and/or transgenic plants.

This is a laboratory suitable for students who are familiar with the basic principles of plant cell structure, tissue culture, sterile technique, and cell transformation (bacterial infection, plasmid vectors, marker genes, selection medium, and enzyme activity assays).


MATERIALS:

(Materials are listed for each group of 2-3 students. Multiply for the number of groups in the class.)

DAY 1 (full period lab)

  1. Nicotiana tabacum (tobacco leaves)

  2. overnight suspension of Agrobacterium tumefaciens (A.t.) containing tumor inducing (Ti) plasmids. The Ti plasmids (pBIRG2) should be engineered to include the B-glucuronidase gene.

  3. sterilized paper punchers and sterilized forceps (scissors can be used instead of punchers)

  4. Bunsen burner

  5. forceps

  6. small beaker of ethanol (80-95%) for flaming forceps and wiping bench area

  7. one 50 ml sterile centrifuge tube with a tight cap

  8. 25 ml 70% ethanol for leaf sterilization

  9. 25 ml 10% Clorox

  10. 150 ml sterile H2O (water can be sterilized by autoclaving in foil-covered flasks)

  11. small piece of sterile filter paper (5-10 cm square)

  12. foil for wrapping Petri dishes

  13. one sterile, empty Petri dish

  14. two sterile Petri dishes containing cocultivation media:
  15. Murashige and Scoogs Basal Salts (Sigma #5519)
  16. 3.0 mg/l Kinetin (Sigma #K0753)
  17. 0.3 mg/l BAP (Sigma # B9395)
  18. 1% agar (10 grams/l)

DAY 3 (15 minutes)

  1. sterile forceps
  2. Bunsen burner and ethanol for flaming forceps
  3. foil for wrapping Petri dishes
  4. two sterile Petri dishes containing selection media:
  5. Murashige and Scoogs Basal Salts
  6. 3.0 mg/l Kinetin
  7. 0.3% mg/l BAP
  8. 400 mg/l carbenicillin (Sigma #C3416)
  9. 20 mg/l benomyl, 50% wetable (available from
  10. garden stores)
  11. 50 mg/l kanamycin (Sigma #K4378)
  12. 1% agar (10 grams/l)

DAY 5 (15-30 minutes, wait 1-3 hours, or overnight, and then 15 minutes again)

  1. X-Gluc histochemical reagent: 10 ml 50 mM phosphate buffer, pH 7.0

      (a) combine:
    • 39 ml 0.2 M NaH2PO4 (31.2 g/l) (Sigma #S9638)
    • 61 ml 0.2 M Na2HPO4 (28.39 g/l) (Sigma #S0876)
      This makes 100ml of a 200 mM phosphate buffer.

      (b) dilute: the 200 mM phosphate buffer 1:4 with distilled H2O

  2. X-GLUC stain
    • dissolve 5 mg 5-bromo-4-chloro-3-indolyl glucuronide(X-gluc) (Sigma #B6650)
      in
    • 100 ml dimethyl formamide (Sigma #D8654)

  3. 1 ml pipettes or transfer pipettes

  4. 24 - well plates (Sigma #M9655)...(2-3 groups can share one plate)

  5. a Petri dish

  6. scapel and sterile forceps (sterile - if you want to keep the leaf disk culture)

  7. dissecting microscope


SAFETY PRECAUTIONS:

Ethanol is flammable. Students should dip the tips of their forceps into the ethanol and then pass them briefly and carefully through the flame. Benches should be clear of all papers and notebooks to reduce the risk of fire should a flaming forceps be dropped. Students should wear goggles.


NOTES TO THE TEACHER

Injured cells on the periphery of each leaf disk release cytoplasm. Some of the chemicals in this cytoplasm stimulate transformation of other cells. The specific mechanism of transformation (wall/membrane weakening etc.) is not clearly understood.

After the β-glucuronidase transformation assay is complete, students can leave the remaining sterile leaf disks on culture media until small tumors of undifferentiated cells (callus) appear on the leaf. These cells can be cultured on growth medium for future experiments.

(Adapted from WWNFF Institute 1993 lab, Dr. Mary Saunders.)


STUDENT INSTRUCTIONS/HANDOUTS: CELL TRANSFORMATION IN TOBACCO LEAF DISKS

INTRODUCTION

In the plant transformation experiment described here, you will see a good model for moving genes into plant cells. By incubating small pieces of tobacco leaf with certain bacterial cells (Agrobacterium tumefaciens) under specific conditions, the bacteria cells will infect the plant tissue and certain plasmid genes will move from the bacteria cells, into the nuclei of plant cells. The plasmid DNA will become part of the plant cells' own DNA. When this happens and the transformed plant cells are regenerated into a plant, the plant is known as a "transgenic" plant.

The bacteria A. tumefaciens is used in this type of transformation because of its natural transforming properties. This bacteria is known to cause crown gall disease in plants. Crown gall disease is characterized by the formation of tumors in plants after infection of wound sites by the bacterium. When infected, masses of undifferentiated embryonic cells are produced and thus form a crown gall tumor. The tumor results from expression of growth hormone genes that have been inserted into the mature plant cells by the bacterial plasmids. These growth hormones are produced in the transformed cells in quantities normally found only in embryonic plant cells.

The crown gall inducing plasmid is a 200 kb plasmid called Ti plasmid (tumor inducing). The particular segment of the Ti plasmid responsible for transformation is the T-DNA (13 kb) which carries genes for plant growth hormones (auxins and cytokinin). When the T-DNA is transferred to a plant, the plant cells begins to produce the growth hormones. An additional section of plasmid DNA controls transformation; the virulence (vir)genes control the transfer of the T-DNA from the plasmid into the plant DNA.

Once this natural model of plant transformation was understood, the Agrobacteriumcould be used to make all different types of transgenic plants. If a scientist wants to produce a plant with a particular gene, he or she adds that gene to the T-DNA sequence in the Ti plasmid and using the Agrobacterium's natural infection mechanism, transgenic plant cells are produced.

In the bacteria that we are using for this experiment, the Ti plasmid (pBIRG2) contains a gene for an enzyme called β-glucuronidase (also called the GUS gene). In this lab, you will be looking for evidence that the GUS gene and thus the DNA from the plasmid containing the GUS gene, has been added to your plant cells. By adding a substrate (X-Gluc) for β-glucuronidase to the dishes containing your transformed cells, you will be able to identify cells that contain the enzyme and therefore must contain the DNA from the plasmid. A blue precipitate is formed at the sites of the enzyme reaction. This blue precipitate will indicate the presence of transformed cells.

METHODS AND MATERIALS:

DAY 1

  1. Wipe your bench area with ethanol before placing any equipment on it.

  2. Obtain a mature leaf. Sterilize the surface of the leaf in the following manner. Take the leaf and place it in a 50 ml sterile tube. Add 25 ml of 70% ethanol to the tube. Make sure the ethanol completely covers the leaf. Lay the tube on its side making sure that the ethanol completely covers the leaf material. Incubate 2-6 minutes. From this point on, forceps should be used for all transfers of the leaf. Forcep tips should be dipped in ethanol and passed through a flame (Bunsen burner) before each leaf transfer. Do NOT hold forceps in the flame; dip the tips only in the ethanol and pass them through the flame. When the flame subsides, they are ready for use.

  3. Pour out ethanol solution into the sink or a waste beaker and replace it with 10% chlorox. Incubate 2-5 minutes. Watch the leaf tissue for any bleaching at the edges. Remove if there is bleaching.

  4. Remove Chlorox® solution and add sterile distilled water to the tube. Incubate for 2-3 minutes.

  5. Repeat the sterile water rinse three times.

  6. Using the sterile forceps and sterile paper punch, punch out about 40 disks from the leaf. Collect the disks in an empty sterile Petri dish.

  7. Using flamed forceps, remove about half of the leaf disks (one by one) from the Petri dish and place them on the first Petri dish containing cocultivation media. Be careful that the culture dish remains sterile! Be careful to keep the culture dish covered and only open it briefly to add a disk, taking care not to contaminate the cover.

  8. Pour about 1.5 ml of Agrobacterium culture (grown overnight) into the Petri dish (without media) holding the remaining disks. Incubate for 5-10 minutes, swirling the plate gently at 2 minute intervals and being careful not to spill the media.

  9. Using flamed forceps, remove leaf disks from the bacterial solution, touch each briefly on a piece of sterile filter paper to remove excess liquid and place the disks on the second Petri dish containing cocultivation media. Make sure that your two co-cultivation dishes (also called plates) are marked clearly so that you know which contains leaf disks that have been exposed to the bacteria.

  10. Wrap the plates completely in aluminum foil and incubate the two dishes for 48 hours at room temperature.

    DAY 3

  11. Use flamed forceps to transfer the disks from the two cocultivation dishes to two Petri dishes containing selection media. Place the leaf disks with the bottom of the leaf facing up, away from the media. Again maintain sterile technique and clearly label the dishes. Wrap the dishes in foil and incubate at room temperature for another 48 hours.

    DAY 5

  12. Fill each well of a 24-well culture plate with 0.5 ml of the X-Gluc histochemical reagent. (Two or three groups will be sharing one of the 24-well plates.)

  13. Using flamed foreceps (if you want to keep the leaf disk cultures), take two leaf disks from one of your selection media dishes and place them in a Petri dish. Cut the disks into quarters. Place one piece of leaf in 6 of the small wells. (You will have two extra leaf disk quarters.) Repeat the procedure with two disks from your other selection dish and place one piece in each of the remaining wells. Discard the extra leaf disk quarters, but do NOT discard your uncut leaf disks.

  14. Incubate the leaf quarters in the X-Gluc reagent at 37°C for 1-3 hours (or overnight if that's more convenient).

  15. When you return, examine the leaf quarters for any blue color. A dissecting microscope might be helpful. Record the number of blue spots located and the number of leaf pieces affected.

  16. Re-wrap your tissue cultures in foil and check them in 3-7 days and again in 7-14 days. If small bubbles begin to appear on the leaf surface, then you have callus (small groups of undifferentiated ) tissue forming. Using sterile technique you can culture the callus on new selection media or other specialized growth media for future observation/ experiments with the transgenic plant tissue.


QUESTIONS:

  1. The major difference between the co-cultivation media and the selection media is the presence of antibiotic and antifungal agents (carbenicillin, benomyl and kanamycin) in the selection media. Why is this selection media critical to the experiment? What problems would you predict it the selection media were not used?

  2. What are all of the controls in this experiment and why is each necessary?

  3. What does the presence of blue color in leaf disk suggest?

  4. Based on your GUS results in two leaf disks, how many of the other leaf disks do you predict have transformed cells in them? Explain your answer and show the calculations.

  5. How could we go about growing up transgenic plants from the transformed cells in this experiment?

  6. Observe the two leaf disk cultures daily for a couple of weeks after the transformation experiment. Record your observations of the disks in both cultures. What evidence is there, if any, confirming that transformation has occurred in the bacteria-exposed plant cells?


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