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Updated 2/7/2016
Floral Dip Protocol for Arabidopsis Transformation
Prepared by Sarah Prewitt, UNT PhD candidate, edited by BGA, adapted from:
Clough SJ and Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. The Plant Journal 16(6): 735-743
Davis AM, Hall A, Millar AJ, Darrah C, Davis SJ (2009) Protocol: Streamlined sub-protocols for floral-dip
transformation and selection of transformants in Arabidopsis thaliana. Plant Methods 5:3
Required Solutions and Materials:
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Arabidopsis plants for dipping grown according to requirements for floral dips
(see below)
LB broth with Rifampycin, Gentamycin and Kanamycin (all at 50 g/mL) (or
other selection, as appropriate)
Dipping solution: 5% Suc, 0.04% Silwet (ie 400 L/L)
Glass petri dish
Spray bottle of distilled H2O
100% humidity chamber for holding plants after dips
Growing Arabidopsis for floral dips:
1)
Fill 3.5 in. square pots with Fafard 3B (or other appropriate) soil mix. Wet thoroughly
with water. The level of wetted potting mix should be even with the edge of the pot.
2)
Distribute seed of appropriate line to the pot. You will want 3 or 4 evenly spaced plants
for dipping. This is easily achieved by distributing 3 or 4 seeds to the four corners of the pot ~2.0
cm (3/4 in) from the sides. Plants are then thinned as the healthiest are identified.
3)
Stratify seeds/pots for 3 days in cold room. Grow plants in a growth chamber with 12 hr
light / 12 hrs dark; 22°C days / 20° nights; ≥ 150 µmoles photons m-2 s-1 for ≥ 28 days. These
conditions will produce robust vegetative growth and strong inflorescences with many closely
packed flowers.
4)
When the 1 inflorescence is ~2.5 cm (1 in) tall, cut to encourage axillary inflorescence
growth; Axillary inflorescence will be ready for dipping 5-8 days after removing 1
inflorescence. The first dip should occur when inflorescences are ~2 to 5 cm long.
Preparing the Agrobacterium:
Day 1: Set up 20 mL cultures in a sterile 250 mL Erlenmeyer flask with a well-isolated single
colony (clonal) of the desired Agrobacterium tumefaciens in LB broth containing Rifampycin,
Gentamycin and Kanamycin (all 50g/mL). We usually use GV3101 mp90 for all plant
transformations; Rif resistance is on the chromosome (GV3101), Gent resistance is on the
disarmed Ti plasmid (mp90), and Kan resistance is on your binary vector – change the selection
for your vector as appropriate.
Hints:
1)
Save some media with antibiotics to use as blank in the spectrophotometer
2)
If you have a lot of cultures, 10 to 15 mL of culture can be grown in 50 mL sterile glass
culture tubes
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Updated 2/7/2016
Grow ~24 hours until O.D.600 is approximately 2.0. Usually a 10X dilution will spec in good
range.
Floral dip procedure:
Day2:
1)
Remove 1-2 mL of Agro culture to a clean microfuge tube for miniprep processing to
ensure plasmid in Agrobacterium is the desired plasmid. Digest plasmid directly or transform
back into E. coli to amplify.
2)
Harvest cells by centrifugation in sterile Oakridge tubes in an SS34 rotor for 10 minutes
at 5,000g, or in 50 mL Falcon tubes in a swinging bucket rotor at 3000 rpm (less effective, but
still gets most of the cells). The rotor should not be chilled, but a chilled rotor probably does not
hurt.
3)
Resuspend the bacterial pellet in 15 mL dipping solution (5% Suc, 0.04% silwet) to a
calculated O.D.600 of 1.0 to 1.5; Pour up to 40 mL of the solution into a glass petri dish
4)
Invert the Arabidopsis inflorescences into the solution such that only the flowers, and as
little vegetative material as possible, are submerged; hold for a few seconds. . It is important to
hold the pots, plants and soil in a way that prevents them from falling into the Agro solution.
5)
Alternatively, if the inflorescences are not long enough for convenient dipping, the tips
can be treated by placing a few drops of Agro solution directly on flowers
6)
After dipping, spritz the plants gently with distilled water and put the plants in a 100%
humidity chamber. This chamber is created spaying the inside of a relatively enclosed space
with little air flow with water (i.e., a garbage bin turned upside down).
7)
Agro strains and pots are generally processed one at a time for greatest viability
(extended exposure of Agro or plants to concentrated Silwet may reduce viability)
8)
Keep dipped plants in the 100% humidity chamber for 24 hrs before returning to the
growth chamber.
9)
Repeat the dips after ~6 days. A third dip can be done as well, ~6 days after that.
10)
Restrain inflorescences to prevent cross-contamination between dipped pots using
bamboo steaks or tubes made from overhead acetate sheets.
11)
Continue watering for ~2 weeks to keep developing siliques healthy, while starting to
reduce water to prevent further flowering, but allowing healthy seed development.
12)
When siliques are mature and have lost chlorophyll, harvest seeds into microcentrifuge
tubes with a small hole in the cap, and dry in a desiccator with Drierite for 7-10 days.
13)
Place the seeds in a zip-loc bag and transfer to -70C for 2 days and then transfer back to
desiccation for 1 day – minimize the amount of condensation that can form directly on the seeds.
14)
Store the seed appropriately and select T1 transgenics.