Protocol for transformation

unknown at dl.ac.uk unknown at dl.ac.uk
Tue Jan 11 13:59:34 EST 1994


To the Arabidopsis net:

A number of people have been requesting a vacuum infiltration
transformation protocol.  I spent a large part of the summer and fall
working on such a protocol, discovering the Bechtold et al. protocol, and
then modifying their protocol.  I had intended to post the most recent
version of my protocol to the network AFTER confirming by molecular means
that all the putative transformants obtained using this most recent
protocol were in fact genuine.  The rate of protocol requests has gone up,
however, and I've decided to go ahead and share this protocol now.  Caveat
emptor. 

We're extremely encouraged that this protocol works and takes the
Arabidopsis transformation business many steps forward in terms of
minimizing the number of person-hours required and in avoiding tissue
culture and regeneration.  My last two experiments gave putative
transformants in about 2/3 of the independent samples (pots with about 12
plants each) attempted.  The overall rate of positives was about one
transformants per 2,500 seed plated for selection. 
Note however that the time required to go from seed to seed is still
months.
Note also that the wisdom of extensive experience has not accrued around
this protocol, i.e., we may all find out a year from now that there are
horrible secondary phenotypes associated with the transformants obtained. 

Thanks to Nicole Bechtold, Jeff Ellis and Georges Pelletier for what looks
to be a very useful new method.

- Andrew Bent
  Staskawicz Lab
  UC-Berkeley   

____________________________________________________________________________


Transformation of Arabidopsis by Vacuum Infiltration

This protocol is based on the work of Nicole Bechtold, Jeff Ellis and
Georges Pelletier.  My modifications were incorporated to streamline their
procedure.  The most significant changes eliminate the need to uproot and
re-plant infiltrated plants.
-  Andrew Bent

Plant Growth:
1.  Grow plants of the appropriate genotype to a stage at which bolts are
just emerging.

I have found that it works well to grow 12-15 plants in a 3.5" pot.  
If pot is covered with nylon window screen after planting, plants grow
through the screen and when pot is inverted for infiltration less dirt
falls out.
If plants are grown for the first four weeks in short days you will get
larger plants and a greater seed yield (transfer plants to long days to
induce bolting).  
Success may also depend on frequent fertilization and strong light
intensity.

2.  Clip off emerging bolts to encourage growth of multiple secondary
bolts.

Infiltration will be done four to eight days after clipping.

Vacuum Infiltration:
3.  Grow a large liquid culture of Agrobacterium carrying the appropriate
construct.

Start a 25 ml overnight (LB + antibiotics) two to three days ahead of time.
 Add this culture to 400ml of LB + antibiotic the day before infiltration.
My experiments were done using A. tumefaciens GV3101.

4.  Harvest cells by centrifugation (5K 10min. in GSA rotor, preferably at
room temp.) and resuspend in 3 volumes infiltration medium (OD600 approx.
0.8).

Harvest cells at an OD600 of >2.0.  A 400 ml culture will give enough cells
for infiltration of at least six pots.

5.  Add Agrobacterium (in infiltration medium) to a dish or beaker and
invert plants (pot, soil, and all) into liquid solution.  Be sure bolts and
entire rosettes are submerged.

A one liter beaker filled with >200 ml of solution fits well with our 3.5"
pots.
Bacterial solution can be extended by reusing for at least one additional
pot.

6.  Place beaker into bell jar.  Draw a vacuum until bubbles form on leaf
and stem surface and solution starts to bubble a bit, then release vacuum
very rapidly.

The necessary time and vacuum pressure will vary lab-to-lab.  Practice on a
few dispensable plants first. Good infiltration is visibly apparent as
uniformly darkened, water-soaked tissue.
Be sure to have good traps in your vacuum system or you will quickly
saturate the pump oil.

7.  Remove plants from beaker, lay them on their side into a plastic flat
and cover with plastic wrap or a dome to maintain humidity.  The next day,
uncover plants and set upright.  

8.  Grow approximately four weeks, keeping bolts from each pot together and
separated from neighboring pots.

Selection of Putative Transformants:
9.  When siliques on plants are very dry, harvest seed (all seed from one
pot together).

For Kanamycin selection:
(Note that Basta selection is much less labor intensive - but your present
binary vector system is more likely to encode antibiotic resistance.)

10.  Pour selection plates.

Plastic 150 x 15 mm petri dishes are convenient.

11.  Sterilize seed.

A variety of sterilization protocols are appropriate.  
I place seed in 15 ml plastic orange cap tubes and then treat:
1 minute in ethanol or isopropanol
5 minutes in 50% Bleach/50% water/0.05% Tween.
3 rinses with sterile water.

It is advisable to add one or two control seeds from a known transformed
plant onto a marked location on at least a few of the selection plates. 
Sterilize these seed also.

12.  Plate seed by resuspending in sterile, room temperature 0.1% agarose
and spreading onto selection plates.  Dry plates in laminar flow hood until
seed no longer flows when plate is tipped.

Use one ml agarose for every 500-1000 seed.  
Plate 2000 to 4000 seed per 150 x 15 mm plate.  Higher densities can make
antibiotic selection less effective.

13.  Vernalize plates for two nights in cold room.  Move plates to growth
chamber.

14.  After about 7 days, transformants should be clearly identifiable as
dark green plants with healthy green secondary leaves and roots that extend
over and into the selective medium.

15.  Transplant plantlets to soil,  grow, and collect seed.

Transplanting success is improved by breaking up agar around root prior to
pulling, by removing any adhering chunks of agar from root before planting,
by saturation of soil with water after transplanting, and by growing plants
under a dome (for high humidity) for the first day or two.  If you break
the root, put plantlet onto a new selection plate for a few days before
transplanting.


Infiltration Medium:					
1/2 X Murashige & Skoog salts				
1 X B5 vitamins						
5.0% Sucrose							
..044 uM Benzylamino Purine		(10 ul per liter of a 1 mg/ml stock in DMSO)

Selection Plates:
1/2 X Murashige & Skoog salts
0.8% Agar
Autoclave, cool, then add:
1 X B5 vitamins
Antibiotic (such as Km 50 ug/ml)

Good Luck!




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