Agrobacterium-Mediated Floral Dip Transformation Protocol

Floral dip is a widely used method for transforming Arabidopsis thaliana by introducing foreign DNA into the plant’s genome. The following is a step-by-step protocol to achieve efficient transformation:

Materials and Reagents:

  1. Arabidopsis thaliana plants at the appropriate stage (5-6 weeks old with actively growing flower buds).

  2. Agrobacterium tumefaciens strain carrying the desired binary vector.

  3. LB medium (Luria-Bertani broth) or YEB medium.

  4. Antibiotics for selection (e.g., rifampicin, gentamicin, kanamycin, or others depending on the strain and plasmid).

  5. 50 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer, pH 5.6.

  6. 5% Sucrose solution.

  7. 0.02% Silwet L-77 or Silwet-408 surfactant.

  8. 1X MS (Murashige and Skoog) medium (for plant growth).

  9. Hormone-free soil mix (e.g., Sunshine mix, Jiffy mix).

  10. Plant growth chamber or growth room with a 16-hour light/8-hour dark cycle.

Equipment:

  1. Sterile 50 mL falcon tubes.

  2. Shaker/incubator.

  3. Centrifuge.

  4. Plant growth chamber or room.

  5. Sterile containers for dipping.

  6. Plastic wrap or humidity dome.

Procedure:

1. Preparation of Agrobacterium Suspension

1.1. Inoculation: Pick a single colony of Agrobacterium tumefaciens carrying the desired plasmid and inoculate into 5 mL of LB or YEB medium containing the appropriate antibiotics. Grow overnight at 28°C with shaking (180-200 rpm).

1.2. Culture Expansion: Next day, transfer 1 mL of the overnight culture into 50-100 mL of fresh LB or YEB medium with the appropriate antibiotics. Grow the culture until the OD600 reaches 0.8-1.0 (usually 4-6 hours).

1.3. Harvesting Cells: Centrifuge the culture at 4,000 x g for 10 minutes at room temperature.

1.4. Resuspension: Discard the supernatant and resuspend the bacterial pellet in infiltration medium (½X MS, 50 mM MES, 5% sucrose, pH 5.6) to a final OD600 of 0.8-1.0.

1.5. Add Surfactant: Add Silwet L-77 to a final concentration of 0.02% (200 µL of Silwet L-77 in 1 L of suspension).

2. Plant Preparation

2.1. Grow Arabidopsis thaliana plants under short-day conditions (8 hours light/16 hours dark) until they have at least 5-6 true leaves. Switch them to long-day conditions (16 hours light/8 hours dark) to induce flowering.

2.2. Ensure plants have a good number of young flowers and flower buds at the time of dipping.

3. Transformation by Floral Dip

3.1. Dipping: Hold the pot with one hand, invert the pot so that the floral clusters are submerged in the Agrobacterium suspension. Gently swirl the plant in the solution for 10-30 seconds ensuring all flowers and buds are fully coated.

3.2. Cover Plants: After dipping, place the plants upright and cover with a plastic wrap or a humidity dome to maintain high humidity for 24 hours. This helps Agrobacterium infection and transformation.

3.3. Incubation: After 24 hours, remove the cover and place plants back into normal growing conditions (16-hour light/8-hour dark cycle).

4. Post-Transformation Care

4.1. Watering: Continue to water the plants normally. Avoid wetting the foliage directly for the first few days post-transformation.

4.2. Seed Set: Allow the plants to set seeds. This process usually takes 3-5 weeks.

5. Harvesting and Screening

5.1. Harvest Seeds: Once the siliques are brown and dry, harvest the seeds by gently shaking the plants over a clean piece of paper or by hand-threshing the siliques.

5.2. Surface Sterilization (Optional): If contamination is a concern, surface sterilize the seeds with 70% ethanol for 1 minute, followed by 10% bleach solution for 10 minutes, and rinse thoroughly with sterile water.

5.3. Selection: Plate seeds on selection medium (e.g., ½X MS agar with the appropriate antibiotic/herbicide). Stratify the seeds at 4°C for 2-4 days in the dark to improve germination.

5.4. Germination and Screening: Transfer the plates to a growth chamber with a 16-hour light/8-hour dark cycle and identify transformants based on selection marker expression (e.g., GFP, antibiotic resistance).

Tips for Successful Transformation:

  • Plant Stage: Ensure plants have young floral buds at the time of dipping as they are more susceptible to transformation.

  • Agrobacterium Quality: Use fresh cultures of Agrobacterium. Old or subcultured bacteria can reduce transformation efficiency.

  • Humidity: Maintain high humidity after dipping to promote Agrobacterium infection.

  • Growth Conditions: Keep a consistent light and temperature regime to avoid plant stress and promote good seed set.