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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:
  2. i)10 ml 50 mM phosphate buffer, pH 7.0
  3. a) combine: 39 ml 0.2 M NaH2PO4 (31.2 g/l)
  4. (Sigma #S9638) 61 ml 0.2 M Na2HPO4
  5. (28.39 g/l) (Sigma #S0876) This makes
  6. 100ml of a 200 mM phosphate buffer.

  7. b) dilute the 200 mM phosphate buffer 1:4
  8. with distilled H2O

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

  10. 1 ml pipettes or transfer pipettes

  11. 24 - well plates (