Simplified Arabidopsis Transformation Protocol
Steve Clough and Andrew Bent, University of Illinois at Urbana-Champaign.
(Brief version for those who are familiar with the method)
Our present protocol (Clough and Bent, 1998; modified from
Bechtold et al. 1993) is extremely simple. We have found that the MS salts,
hormone, etc. make no difference, that OD of bacteria doesn't make much
of a difference, that vacuum doesn't even make much of a difference as
long as you have a decent amount of surfactant present. Plant health is
still a major factor - healthy fecund plants make a big difference! With
this method you should be able to achieve transformation rates above 1%
(one transformant for every 100 seed harvested from Agrobacterium-treated
For higher rates of transformation, plants may be dipped two
or three times at seven day intervals. We suggest one dip two days after
clipping, and a second dip one week later. Do not dip less than 6 days
Grow healthy Arabidopsis plants until they are flowering. Grow under long
days in pots in soil covered with bridal veil, window screen or cheesecloth.
(optional) Clip first bolts to encourage proliferation of many secondary
bolts. Plants will be ready roughly 4-6 days after clipping. Clipping can
be repeated to delay plants. Optimal plants have many immature flower clusters
and not many fertilized siliques, although a range of plant stages can
be successfully transformed.
Prepare Agrobacterium tumefaciens strain carrying gene of interest
on a binary vector. Grow a large liquid culture @ 28oC in LB
with antibiotics to select for the binary plasmid, or grow in other media.
You can use mid-log cells or a recently stationary culture.
Spin down Agrobacterium, resuspend to OD600 = 0.8 (can be higher
or lower) in 5% Sucrose solution (if made fresh, no need to autoclave).
You will need 100-200 ml for each two or three small pots to be dipped,
or 400-500 ml for each two or three 3.5" (9cm) pots.
Before dipping, add Silwet L-77 to a concentration of 0.05% (500 ul/L)
and mix well. If there are problems with L-77 toxicity, use 0.02% or as
low as 0.005%.
Dip above-ground parts of plant in Agrobacterium solution for 2 to 3 seconds,
with gentle agitation. You should then see a film of liquid coating plant.
Some investigators dip inflorescence only, while others also dip rosette
to hit the shorter axillary inflorescences.
Place dipped plants under a dome or cover for 16 to 24 hours to maintain
high humidity (plants can be laid on their side if necessary). Do not expose
to excessive sunlight (air under dome can get hot).
Water and grow plants normally, tying up loose bolts with wax paper, tape,
stakes, twist-ties, or other means. Stop watering as seeds become mature.
Harvest dry seed. Transformants are usually all independent, but are guaranteed
to be independent if they come off of separate plants.
Select for transformants using antibiotic or herbicide selectable marker.
For example, vapor-phase sterilize and plate
40 mg = 2000 seed (resuspended in 4 ml 0.1% agarose) on 0.5X MS/0.8% tissue
culture Agar plates with 50 ug/ml Kanamycin, cold treat for 2 days, and
grow under continuous light (50-100 microEinsteins) for 7-10 days.
Transplant putative transformants to soil. Grow, test, and use!
Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium-mediated
gene transfer by infiltration of adult Arabidopsis thaliana plants.
C. R. Acad. Sci. Paris, Life Sciences 316:1194-1199.
Clough SJ and Bent AF, 1998. Floral
dip: a simplified method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant J 16:735-43.
Additional commentary can be found by searching the Arabidopsis newsgroup
NOTE: Obtain proper approval for
transformation work from institutional authorities. Autoclave and properly
dispose of all materials.