Molecular Techniques and Methods

Agrobacterium-Mediated Transformation
of Arabidopsis by Vacuum Infiltration

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Infiltration Medium (1 liter)
0.5 x MS Salts (Gibco) -------------------------------- 2.15 g
B5 Vitamins -------------------------------------------- 1 ml
5% Sucrose -------------------------------------------- 50 g
0.044 uM Benzylamino purine --------------------------
0.03% Silwet L-77 (OSI Specialty) --------------------- 0.3 ml
Add deionized H2O to make a final volume of --------- 1 liter


B5 Vitamins (100 ml)

Nicotinic acid 0.1 g
Thiamine-HCl 1 g
Pyridoxine-HCl 0.1 g
myo-Inositol 10 g
  • Add deionized H2O to make a final volume of 100 ml.
  • Store at -20oC.


    Sterile 0.1% Agarose (100 ml)
    0.1% Agarose ------------------------------------------ 0.1 g
    Deionized H2O ----------------------------------------- 100 ml
  • Autoclave.


    Selection Medium (1 liter)
    1 x MS Salts (Gibco) ---------------------------------- 4.3g
    0.3% Sucrose ------------------------------------------ 3 g
    1-1.5% Phyta agar ---------------------------------------- 10-15 g
    Kanamycine -------------------------------------------- 50 mg
    Timentin ------------------------------------------------ 200 mg
    Add deionized H2O to make a final volume of --------- 1 liter
  • Autoclave.




    PROCEDURES

    Plant Growth

    1. Plant Arabidopsis seeds on top of cheesecloth-covered soil in pot.
  • Use rubber band to secure the cheesecloth.


  • 2. Vernalize the seeds for 2-3 days in 4oC cold room.

    3. Move pots to growth chamber (24oC and 16 hour-photoperiod).

    4. After 18-21 days, clip off the primary bolt to encourage the growth of several secondary bolts.

    5. Perform transformation in 3-4 days after clipping.
  • Do not wait until floral buds open!



  • Transformation by Vacuum Infiltration

    6. Grow 10 ml culture of Agrbacterium in LB and antibiotics for 2 days at 28oC.

    7. Inoculate 1 ml Agrbacterium culture to a 100 ml of LB medium with antibiotics.

    8. Incubate overnight at 28oC.

    9. Harvest cells by centrifugation at 3,000g for 10 minutes.

    10. Resuspend Agrobacterium in Infiltration Medium to an OD600 0.8-1.2.

    11. Invert pot and put floral buds into the Infiltration Medium.

    12. Put into vacuum for 15 minutes at 13-15 in3Hg.

    13. Remove pot from the vaccumm and lay it on its side in tray to remove excess bacterial solution from the pot.

    14. Immediately cover the tray with plastic wrap to prevent from drying.

    15. Put tray in growth chamber (24oC and 16 hour-photoperiod).

    16. After 4-6 days, remove plastic cover and stand pots upright position.

    17. Grow 3 weeks in growth chamber (24oC and 16 hour-photoperiod).


    Selection of Transformants

    18. Start collecting seeds from transformed plants at 3rd week.
  • Collect seeds again at 4th week.
  • Collect seeds again at 5th week.
  • (Usually the first batch of seed collection has the highest ratio of transgenics.)



  • 19. Sterilize seeds in 16 ml centrifuge tubes with;
  • 70% ethanol for 10 minutes.
  • 50% Bleach and 3% Tween 20 for 15 minutes.
  • Rinse with sterile H2O three times.


  • 20. Resuspend seeds in 5-7 ml of sterile 0.1% agarose.
  • Care should be taken to prevent fungal contamination.


  • 21. Spread seeds on the Selection Medium in petriplates (15 x 150 mm).
  • Put 2,000-4,000 seeds per plate.
  • (50,000 seeds/ 1 g)


  • 22. Vernalize seeds for 2-3 days in cold room.

    23. Move petriplates to growth chamber (24oC and 16 hour-photoperiod).

    24. After 7-10 days, transformants should be visible as dark green plants with roots, which extend over and into the Selection Medium.

    25. Transfer transformants onto the new Selection Medium in petriplates.

    26. Grow additional 1 week in growth chamber (24oC and 16 hour-photoperiod).

    27. Transfer (kanamycine-resistant) transformants into soil.


    NOTES


    KIT INFORMATION


    REFERENCES


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

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified