Molecular Techniques and Methods

Agrobacterium-Mediated Leaf Disc Transformation

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Surface-sterilized leaf discs or other axenic plants are infected with the appropriate strain of A. tumefaciens, and cocultured on regeneration medium for 2 or 3 days. During this time, the virulence genes in the bacteria are induced, the bacteria bind to the plant cells around the wounded edge of the explant, and the gene transfer process occurs. After the transformation has occurred, the explants are transferred to regeneration/selection medium. This contains 500 ug/ ml carbenicillin to kill the bacteria and the appropriate antibiotics (usually kanamycin) to inhibit untransformed plant cells. During the next 3-4 weeks, the transformed cells grow into callus or differentiate into shoots via organogenesis. Between 3 and 6 weeks, the shoots develop enough to remove them from the explant and induce rooting in preparation for transfer to soil.




MATERIALS AND SOLUTIONS

MS-G Medium (1 liter)
MS Salts (Gibco) ----------------------------------- 4.3 g
B5 Vitamin Stock ---------------------------------- 1 ml
3% Sucrose ---------------------------------------- 30 g
0.8% Agar ------------------------------------------ 8 g
Add deionized H2O to make a final volume of ----- 1 liter
  • Adjust the pH to 5.7 with NaOH.


    B5 Vitamin Stock (100 ml)
    myo-Inositol ---------------------------------------- 10 g
    Thiamine-HCl --------------------------------------- 1 g
    Nicotinic acid --------------------------------------- 0.1 g
    Pyridoxine-HCl ------------------------------------- 0.1 g


    MS-H Medium (1 liter)
    MS-G Medium ------------------------------------ 1 liter
    Benzyladenine (BA) -------------------------------- 1 mg
    Napthaleneacetic acid (NAA) ---------------------- 0.1 mg


    MS-S Medium (1 liter)
    MS-H Medium ------------------------------------- 1 liter
    Carbenicillin ---------------------------------------- 500-1,000 mg
    Kanamycin ----------------------------------------- 100 mg


    MS-R Medium (1 liter)
    MS-G with 0.6% Agar ----------------------------- 1 liter
    Carbenicillin ---------------------------------------- 500 mg
    Kanamycin ----------------------------------------- 100 mg




    PROCEDURES

    1. Sterilize seeds by soaking in;
  • 70% ethanol for 15 minutes.
  • 10% bleach solution (containing 0.1% Tween 20) for 30 minutes.
  • Three rinses in sterile H2O.


  • 2. Germinate sterile seeds on MS-G Medium and grow in growth chamber until cotyledons are fully expanded.

    3. Remove cotyledons by cutting at their petiole, and then cut in half to increase the wounded edge.

  • (Alternatively, grow seedlings in flats under moderate light and temperature and low humidity to produce uniform, healthy plants of small size. Start new flats each week or two and discard older plants.

  • Harvest healthy, unblemished leaves from young plants and sterilize in
  • 10% bleach solution (containing 0.1% Tween 20) for 15 to 20 minutes with gentle agitation.
  • Rinse 3 times with sterile H2O.

  • Cut into small strips or squares to produce a wounded edge.)



  • 4. Preculture explants for 1 or 2 days upside down on MS-H Medium to allow initial growth and to eliminate those that were damaged during sterilization or handling. Take only the best explants.

    5. Grow A. tumefaciens culture overnight in LB with appropriate antibiotics. Prepare the culture for inoculation of explants by taking an overnight culture and diluting 1 to 10 with MS-G Medium (1 X 109 cells/ml).

    6. Inoculate explants by immersion in the culture of A. tumefaciens. Be careful not to over-soak the tissue. Blot dry gently as soon as all wounded edges have contacted the inoculum.

    7. Place explants upside down on MS-H medium and incubate for 2-3 days.

    8. Transfer explants to MS-S Medium and incubate for several weeks.

    9. After 2-3 weeks cut explants to separate clearly independent sites of transformation and transfer to fresh MS-S Medium.

    10. Transfer entire explants to MS-R Medium when shoots appear, even if not suitable for removal of individual shoots from the explant.

    11. When defined stems are visible, cut cleanly from the explant and callus and place upright in MS-R Medium to root. Take only one shoot from each explant to ensure no siblings are propagated.

    12. Before removing rooted shoots from sterile culture, transfer a leaf to selection medium to test for resistance to kanamycin. This will later provide information on continued expression of the vector genes in the plants.

    13. When roots first appear, remove plantlets, wash agar from base and plant in sterile soil.

    14. After 7 to 10 days, slowly crack open and back off the lid to reduce the humidity gradually until plants are acclimated to the ambient humidity.

    15. Fertilize and grow under standard plant growth conditions.




    NOTES

  • Uniform, clean, and succulent young plants will perform best. it is worthwhile to take care and effort to ensure a steady, healthy supply of plants is always at hand.

  • Be very gentle since the bleach will damage wounded leaves. Plants from a dirty environment may have internal contaminates that bleach can never remove. Leathery leaves or very weak, etiolated leaves may be damaged by-the bleach. Damaged tissue should not be used.

  • Discs provide a very uniform explant while strips or squares are easier to produce in large quantities. Avoid excessive wounding during the process.

  • Preculturing is highly recommended since it increases the efficiency of transformation and permits rejection of unhealthy explants before inoculation.

  • It is prudent to start cultures 2 days before inoculation to confirm the viability of the culture a day early.

  • The idea is to inoculate the edge rather than soak the internal tissues with bacteria.

  • Longer coculture times will boost transformation but can also result in more bacterial overgrowth later during selection.

  • The rooting on medium containing kanamycin serves to screen for transgenic plants that continue to express the T-DNA.

  • Gradual reduction in the humidity is necessary to harden off the plantlets in soil and can be controlled by gradual opening of the lid. The roots must develop and the leaves must develop a protective wax cuticle. If the plantlets die from fungal contamination, the lid should be opened faster. If the plantlets die from wilting, the lid should be opened slower.



  • KIT INFORMATION


    REFERENCES

  • Deak, M., Kiss, G.B., Koncz, C. and Dudits, D., 1986, Transformation of Medicago by Agrobacterium mediated gene transfer. Plant Cell Rep 5: 97-100.

  • DeBlock, M., Herrera-Estrella, L., Van Montagu, M., Schell, J. and Zambryski, P., 1984, Expression of foreign genes in regenerated plants and their progeny. EMBO J. 3: 1681.

  • Fraley, R.T., Rogers, S.G., Horsch, R.B., Eichholtz, D.A., Flick, J.S., Fink, C.L., Hoffmann, N.L. and Sanders, P.R., 1985, The SEV system: a new disarmed Ti plasmid vector for plant transformation. Bio/Technology 3: 629-635.

  • Hood, E.E., Fraley, R.T., Chilton, M.D., 1987, Virulence of Agrobacietium tumefaciens strain A281 on legumes. Plant Physiol 83: 529-534.

  • Horsch, R.B., Fraley, R.T., Rogers, S.G., Sanders, P.R., Lloyd, A. and Hoffmann, N., 1984, Inheritance of functional foreign genes in plants. Science 223: 496-498.

  • Horsch, R.B., Fry, J., Hoffmann, N.L., Wallroth, M., Eichholtz, D., Rogers, S.G. and Fraley, R.T., 1985, A simple and general method for transferring genes into plants. Science 227: 1229-1231.

  • Lloyd, A.M., Barnason, A.R., Rogers, S. G., Byrne, M.C., Fraley, R.T. and Horsch, R.B., 1986, Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens using a gene conferring hygromycin resistance. Science 234: 464-466.

  • McCormick, S., Niedermeyer, J., Fry, J., Barnason, A., Horsch, R., and Fraley, R., 1986, Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacietium tumefaciens. Plant Cell Rep 5: 81-84.

  • Shahin, E. and Simpson, R., 1986, Gene transfer system for potato. Hort. Sci. 21: 1199-1201.


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