Infiltration protocol

Isabelle Carre ic2m at FARADAY.CLAS.VIRGINIA.EDU
Sun Nov 27 20:49:23 EST 1994


Dear Netters,

A few people recently asked me for my protocol for
transformation of Arabidopsis by vacuum infiltration...
and I just found out that what I sent out contains a 
dreadful typo, that probably will make the transformation
unsuccessful.

You need to infiltrate the plants under 400 mm Hg, Not 40!


Here is the revised version:


                                                                
Transformation of Arabidopsis by vacuum infiltration . 
Isabelle Carr
(modified from: Bechtold N., Ellis J., and Pelletier G. (1993) 
C.R. Acad. Sci. Paris, 316: 1194-9)

We used the Arabidopsis ecotype Landsberg (erecta) and the 
agrobacterium strain ABI in these experiments.

Growth of plants

Sow Arabidopis in large flats. Thin to less than 1 plant per 
square inch after 2-3 weeks. Water every 2 weeks with 0.5 X 
Hoaglands solution. As the plants start to bolt, cut the
primary inflorescence to encourage the development of several
inflorescences from axillary buds. Allow these to grow 2-5 cm 
tall. Make sure that the plants are watered abundantly the day
before infiltration, so that their stomates are wide open.

Agrobacterium culture

Day -5. Streak agrobacterium from a glycerol stock onto an LB
plate (with appropriate antibiotics). Grow at 24-28 Cfor 48 h.

Day -3. Start a 5 ml preculture from a single colony, in 5 ml
of LB (+ antibiotics) at 24-28 C.

Day -2. Start a 1 l culture with 1 ml of the preculture (in LB
+ antibiotics)

D-Day. Spin down the culture (3000 rpm, 10 min). Discard the 
supernatant. Resuspend the cells in 200 ml of infiltration 
medium (MS + 5 % sucrose, 10 ug/l Benzyl amino purine). 
Transfer to a 1-l beaker.

Infiltration

I routinely infiltrate 20 plants per construct. This yelds up 
to 55 transformants on a good day!

Uproot 20 plants: lift the plant with its surrounding soil 
using a pair of forceps.  Place upright on a tray filled with 
water. The soil falls off, and leaves clean, intact roots.

Transfer 10 plants to the agro suspension. I try to put them 
in upside down, so that the inflorescences and less developped 
axillary buds come in contact with the bacteria.  Transfer 
the beaker to a vacuum dessicator.

Turn on the vacuum. Infiltrate 10 min at 400 mm Hg. Longer 
will cause a lot of mortality. 

Release the vacuum as rapidly as possible.  Transfer the plants
to individual pots (I use the Arasystem). 

The same agro suspension can then be used to infiltrate the 
remaining plants.  It looks messy, but it works!

Selection

Collect seed from individual plants (using aracons).  Sterilize, 
and plate individual seed batches on 200 x 20 mm plates.

Selection medium:       MS + 3 % sucrose
                        Kanamycin (50 mg/l) or other antibiotic 
                        for selection
                        Carbenicillin (100 mg/l) to prevent 
                        growth of agrobacterium.

Transfer resistant seedlings to soil as soon as they make 
primary leaves.






Isabelle Carr
Post-Doc (Steve Kay's Lab)
Dept of Biology
University of Virginia
Charlottesville, VA 22902

E-mail: ic2m at faraday.clas.virginia.edu



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