Molecular Techniques and Methods

Agrobacterium-Mediated Transformation
of Tobacco Leaf Disc

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Leaf disc transformation of tobacco is the paradigm for Agrobacterium-Mediated transformation of plant tissues and subsequent selection and regeneration of transgenic plants. This system permits efficient gene transfer, selection and regeneration to be coupled together in a simple process. Tobacco is an excellent host for A. tumefaciens, and also responds exceedingly well in culture.


MATERIALS AND SOLUTIONS

MS 30 Liquid Medium (1 liter)
MS salts (Gibco) --------------------------------------- 4.32 g
3% Sucrose -------------------------------------------- 30 g
  • Adjust pH to 5.7-5.8 with NaOH.
  • Autoclave.


    MS 30 Solid Medium (1 liter)
    MS salts (Gibco) --------------------------------------- 4.32 g
    3% Sucrose -------------------------------------------- 30 g
    0.8% Agar --------------------------------------------- 8 g
  • Adjust pH to 5.7-5.8 with NaOH.
  • Autoclave.


    MS 15 Liquid Medium (1 liter)
    MS salts (Gibco) ---------------------------------------- 4.32 g
    1.5% Sucrose ------------------------------------------- 15 g
  • Adjust pH to 5.7-5.8 with NaOH.
  • Autoclave.


    Regeneration Medium (1 liter)
    Autoclaved MS30 Solid Medium ------------------------ 1 liter
    0.2 um Filter-Sterilized BA (Benzyl Amino purine) --------- 2 mg
    (Optional) 0.2 um Filter-Sterilized NAA ----------------------- 1 mg
    0.2 um Filter-Sterilized Kanamycin -------------------------100mg
    0.2 um Filter-Sterilized Cefotaxime (or, Carbenicilline) ----- 500mg


    Selective Medium (1 liter)
    Autoclaved MS30 Solid Medium ------------------------- 1 liter
    0.2 um Filter-Sterilized Kanamycin -------------------------100mg
    0.2 um Filter-Sterilized Cefotaxime (or, Carbenicilline) ----- 500mg
    0.2 um Filter-Sterilized Timentin ------------------------------ 500mg




    PROCEDURES

    1. Sterile tobacco seeds as follow:
  • Sterilize in 70% ethanol for 15 minutes.
  • Sterilize in 50% bleach for 15 minutes
  • Rinse with sterile H2O three times.


  • 2. Germinate seeds in sterile magenta jar containing MS 30 Solid Medium.

    3. Grow tobacco plants in sterile condition for 1-2 months, or until fully expanded green leaves obtained.

    4. Collect fully expanded, young leaves (2nd-6th leaves) in sterile condition.

    5. Cut the leaves into MS 15 Liquid Medium in petri plates.
  • Avoid using the main vein in the older leaves.


  • 6. Rsuspend overnight grown Agrobacterium culture in MS15 Liquid Medium (0.4-0.6 A595nm).

    7. Transfer the leaves into petri dish containing the MS 15 Liquid Medium and overnight grown Agrobacterium culture.

    8. Poke the leaves with forceps and blade.

    9. Leave for 10 minutes with occasional agitation.

    10. Blot the leaves between 2 sheets of sterile Whatman filter paper.
  • Do not use sterile paper towel, which causes fungal contamination occasionally.

    11. Place leaves on MS 30 Solid Medium for co-cultivation for 1-2 days in growth chamber.

    12. Transfer the leaves to a Regeneration Medium.

    13. Put in growth chamber exposed to light for 3-4 weeks.
  • (Transfer to Regeneration Medium every 7 days).


  • 14. When regenerated shoots appear (1.5cm) cut them out with sharp scalpel.

    15. Transfer to Selective Medium and check for rooting.
  • If transformed, roots will appear within 2-4 weeks depending on gene constructs.


  • 16. Select transformed plants, cut to multiply, and transfer into magenta jars containing Selective Medium.

    17. Transfer plants to soil.

    18. Check transformants by PCR, northern analysis, RT-PCR, and WISH.


    NOTES


    KIT INFORMATION


    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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