Molecular Techniques and Methods

Agrobacterium-Mediated Transformation
of Alfalfa Leaf Disc

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Certain alfalfa genotypes (e.g., Regen-SY) are more amenable to regeneration than others. However, even within same cultivar there is much variability with regard to regenerability. Use a clone from a single plant that has been found to be highly regenerable. Agrobacterium tumefaciens LBA4404 is used to transform alfalfa leaf discs. Anytime a plant transformation is done, an untransformed control (e.g., no Agrobacterim) should be done as well, to be sure that any untransfonned embryos are killed in antibiotic-containing medium.




MATERIALS AND SOLUTIONS

B5h Medium for Plant Cell Culture (1 liter)

NaH2PO4.H2O 0.15 g
KNO3 33 mg
(NH4)2SO4 0.134 g
MgSO4.7H2O 0.5 g
FeSO4.7H2O 27.8 mg
EDTA-Na2 37.3 mg
Proline 0.5 g
Sucrose 20 g
Calcium Chloride Stock 1 ml
Micronutrients 1 ml
Potassium Iodide Stock 1 ml
2,4-D Stock 1 ml
Kinetin Stock 0.1 ml
B5 Vitamins 1 ml
Phytagar 8 g

  • Adjust pH to 5.7 with 1 M KOH.
  • Autoclave.
  • Add 30 ml B5h Stock Aminos and any antibiotics.


    Micronutrients (100 ml)

    MnSO4.H20 1 g
    H3BO3 0.3 g
    ZnSO4.7H20 0.2 g
    Na2MoO4.2H20 25 mg
    CuSO4.5H20 2.5 mg
    CoCl2.6H20 2.5 mg

  • Add deionized H2O to make a final volume of 100 ml.
  • Store at 4oC.


    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.


    Calcium Chloride Stock (100 ml)
    CaCl2.2H20 ----------------------------------------- 15g
    Add deionized H2O to make a final volume of ------ 100 ml
  • Store at 4oC.


    Potassium Iodide Stock (100 ml)
    KI --------------------------------------------------- 75mg
    Deionized H2O -------------------------------------- 100 ml
  • Store in dark bottle at 4oC.


    B5h Stock Aminos (500 ml)

    L-Glutamine 13.3 g
    Serine 1.66 g
    Adenine 8.4 mg
    L-Glutathione 0.17 g
  • Add deionized H2O to make a final volume of 500 ml.
  • Filter sterilize and store at 4oC in 30 ml aliquots.


    2, 4-D Stock (1 mg/ ml)
    Dissolve 100 mg 2,4-D in 1 ml 1N KOH, add hot water to 100 ml.


    Kinetin Stock (1 mg/ ml)
    Dissolve 20 mg Kinetin in 1 ml 1M HCl, bring to 20 ml with water




    MS (Murashige and Skoog) Medium (1 liter)

    MS Basal Salts (Gibco) 4.3 g
    1000 x Nitsch's Modified Vitamins 1 ml
    Sucrose 20-30 g
    myo-Inositol 100 mg
    Phytagar 5 g
  • Add deionized H2O to make a final volume of 1 liter.
  • Adjust pH to 5.8 with NaOH.


    1000 x Nitsch's Modified Vitamins (200 ml)

    Glycine 400 mg
    Nicotinic acid 300 mg
    Pyridoxine-HCl 100 mg
    Thiamine-HCl 100 mg
    Folic acid 100 mg
    Biotin 50 mg
  • Add deionized H2O to make a final volume of 200 ml.




    PROCEDURES

    1. Take young green trifoliates from alfalfa Regen-SY plants. These leaves should be expanded, and only the first or second fully expanded leaf from the shoot apex. Use small leaves. It is best to use leaves from vigorously growing stem that have been cut back 2 to 3 weeks previously. Use only perfect looking leaves - no visible infections or insect damage.

    2. Surface sterilize leaves by rinsing
  • 15 seconds in soapy water,
  • 15 seconds in 70% ethanol,
  • 1 minutes in 20% Chlorox bleach + 0.1% Tween-20,
  • Rinse three times in sterile distilled H2O.

  • All these manipulations can be done in a sterile petri dish and solutions poured off by opening the lid just a crack. Float the trifoliates on sterile water in a petri dish.

    3. Cut leaves in rectangular shape by razor blade in hood and immediately put the leaves on solid B5h (h is for hormones) plates. Wounding the leaves further by pressing down in the center of the leaf blades with the ends of the forceps a couple of times seems to increase transformation efficiency. Remember, Agrobacterium only infects those cells imnidiately adjacent to a wound site.

    4. When sufficient leaf explants are available, proceed with transformation. Because bacterial and fungal contamination is common, it is best to put no more than 10-12 leaf disks on a plate and do many plates (10 or more) so that contaminated ones can be thrown out.

    5. Add 1 ml of Agrobacteriun strain LBA4404 containing the binary vector (grown overnight in YEP media with appropriate antibiotics, early to mid log phase culture may be best) to 10 ml of liquid MS or B5h medium in a petri plate. Float explants on top of medium and let infect for 30 minutes.

    6. Remove each explant and remove as much liquid as possible on the sterile filter paper.

    7. Put them back on solid B5h plates.

    8. Incubate in growth chamber for 4 days (16 hour light, 24oC).

    9. Wash explants in 2 changes of sterile water to remove Agrobacteria. Float them in a petri dish with sterile water, then with a small transfer spatula, gently scrape off most visible bacterial growth from each one in the water and transfer to a fresh plate of water. No visible bacterial growth should remain on the explants following washing. Be sure to have the explants in the water the minimum time possible to get the bacteria adequately washed off.

    10. Put them back on new B5h plates and incubate in growth chamber for another 3 days. Wash each plate separately.

    11. Wash explants two times with sterile distilled H2O. The third petri dish of water should have 10 mg/ ml Timentin and 12.5 mg/ml Cefataxime. Soak the explants 10-15 minutes in this third petri dish.

    12. Put explants onto solid B5h plates that have 500 mg/L timentin and 25 mg/L kanamycin (for pBI101 and pKYLX71 vectors) .

    13. Incubate plates in growth chamber.
    Some bacteria that reside in the leaves just are not killed by the surface sterilization and are quite resistant to both timentin and kanamycin. If that should be a problem cefotaxim works quite well. Make a solution of 250 mg/ml cefotaxim and spread 50-250 ul of this on the plates just before putting on the explants. Be aware that the Cefataxime is quite toxic to the plant tissue also, but they tolerate short exposures to it quite well. However, once visible bacterial growth is detected, it is best to discard the explant and transfer any uncontaminated explants onto a new plate.

    14. After 2 more weeks leaf edges should have formed callus and turned yellowish in color. Occasionally small green spots can be seen that represent the globular stage of the developing somatic embryos.

    15. Move calli and embryos to B5 plates with antibiotics (100 mg/L timentin and 25 mg/L kanamycin) but without hormones.

    16. After another two weeks, transfer to new plates (B5 with antibiotics; 100 mg/L timentin and 25 mg/L kanamycin). There should be a lot of embryos (more than 20 per explant) at this stage.

    17. Spread calli to expose embryos that are hidden, making sure that calli from different explants do not mix. (There could be a lot of embryos at the bottom where the calli were in contact with the media). Even untransformed control explants may make embryos by this stage, but if they are spread out so they come in direct contact with the medium they should soon turn yellow and die.

    18. Let embryos develop for one more week. Some of them should look like little torpedos at this stage.

    19. Take individual, developed embryos and put them on B5 plates with antibiotics (100 mg/L timentin and 25 mg/L kanamycin).

    20. Plantlets are formd after two or three weeks and occasionally will form roots also. Transfer plantlets after real leaves are visible to Magenta boxes with MS media with 25 mg/L kanamycin and 100 mg/L timentin.

    21. Plants can be propagated by cutting, and once they have rooted they can be put into soil.




    NOTES




    KIT INFORMATION




    REFERENCES

  • Woo, HH, Orbac, M, Hirsch, AM, Hawes, MC (1999) Meristem-localized inducible expression of a UDP-Glycosyltransferase gene is essential for growth and development in pea and alfalfa, The Plant Cell. 11 (12): 2303-2316.


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