Transformation by vacuum infiltration--II

Steven J. Clough sjclough at uiuc.edu
Fri Jul 4 10:35:28 EST 1997


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Several people asked that we post a summary of the poster on the
Agrobacterium vacuum-infiltration transformation method that we
presented at the 8th International Arabidopsis Meeting, Madison,
Wisconsin, June 25-29, 1997.


A few notes concerning the poster and this message:

-- The main differences between this new protocol and protocols posted
in the past by N. Bechtold, A. Bent, T. Arashi, the Green Lab, and
others, is that we find we can eliminate the MS salts and BAP and most
importantly, can eliminate the vacuum infiltration.

-- We decided to present the poster a few days before the meeting,
hence there was no abstract in the meeting book.

-- Our title was "Transformation by vacuum infiltration with
Agrobacterium--the vacuum (and other factors) are not necessary."  We
want to emphasize, however, two key points:

1)  We were not the first to notice that vacuum is not necessary. In
fact, there have been several researchers commenting on obtaining
transformants by dipping plants and some of these people posted their
results on the Arabidopsis Bio-Net many months ago.

2)  At this point it is not clear if we have worked out a 'better
method'. Our transformation rates were actually a few-fold lower using
the dip as opposed to the vacuum infiltration protocol.  What the new
protocol does do is streamline the process and further reduce labor
inputs, which may on balance be preferable despite possibly lower
transformation rates.


We plan to prepare a more complete report of these results for
publication at some point in the near future, possibly in Weeds World.
In the meantime, we hope this posting will be of use to the community.



Best wishes,


Steve Clough and Andrew Bent




____________________________

Summary of Poster


Poster Title: Transformation by vacuum infiltration with
Agrobacterium--the vacuum (and other factors) are not necessary.


Goals of the project:

   -Simplify / improve infiltration protocol.

   -Determine which components are essential for successful
transformation.

   -Use what we learn to attempt similar transformation of other plant
species.


Standard inoculation medium that we use in our lab:

        1/2 strength MS medium with vitamins (we use Sigma M-5519)

        5%  sucrose

        44 nM  Benzylaminopurine (BAP)

        0.005%  Silwet L-77 (can be purchased from Lehle seed at
http://www.arabidopsis.com)


In all experiments (except Expt. #1), we used Arabidopsis ecotype Col-O
and A.  tumefaciens strain GV3101 (Koncz, C. and J. Schell, Mol. Gen.
Genet. 204:383-396).  We planted 12-16 plants per 3 1/2 inch pot and
pooled the seed from each pot for selection on  kanamycin plates to
determine % transformation.


More detailed notes on our older "standard procedure" can be found at:

http://www.bio.net:80/hypermail/ARABIDOPSIS/9312/0083.html

A number of related protocols with subtle modifications and comments
can be located by scanning the BioSci newsgroup archives of the
Arabidopsis newsgroup.  This information is not essential to your
effort, but if you are interested you can look on the WWW under:

http://genome-www.stanford.edu/cgi-bin/biosci_arabidopsis.  See also
http://www.bch.msu.edu/pamgreen/green.htm.


All transformation rates are expressed as Percent Antibiotic-Resistant
Seedlings harvested from originally treated plant:

  (#Km-resistant seedlings/Total # seed plated)x(100)


All experiments were done with at least 2 replications within the
experiment; however, not all experiments have been repeated.  Also, all
of these results are from greenhouse grown plants that did not look
ideal in terms of health. We actually prefer to use very healthy,
growth-chamber grown plants.


We are in the process of further testing, so the following data should
be regarded as "preliminary results" or "work in progress."


Expt. 1:    Effect of vacuum on A. tumefaciens strain EHA105.

We found subjecting bacteria to a vacuum for time periods between 15
sec and 10 min (which led to freezing of the bacterial suspension) did
not significantly lower the viable cell count.


Expt. 2:     Pre-inducing Agro for vir gene expression did not seem to
be important:

Grew Agro on CIB induction plates (pH 5.5, 200 uM Acetosyringone, low
phosphate, Fullner,K.J., K.M.Stephens, E.W.Nester.
Mol.Gen.Genet.245:704-715) at 16-18 C. Scraped up cells and suspended
in standard inoculation medium. Infiltrated by vacuum. Transformation
rates: 0.05 and 0.18%.

Also tried growing cells in liquid LB cultures and then adding 100 uM
Acetosyringone to inoculation medium: 0.5, 0.4, 0.27% transformants.

Controls (std. protocol): 0.82, 0.36% transformation.

(This expt. has NOT been repeated, note also that pre-induction was on
plates and not liquid).


Expt. 3:     MS medium does not seem to be important:

Replaced MS medium with 10 mM MgCl2: 0.5, 0.55, 1.13, 0.25%
transformants.

Controls: 0.48, 0.56, 0.82, 0.38% transformants.

(This experiment has been repeated).


Expt. 4:     BAP does not seem to be very important, but it might have
a slight effect.  Too much BAP appears to be deleterious:

Standard inoculation medium minus the BAP: 0.29, 0.133% transformants.

44 uM BAP instead of 44 nM: 0.03, <<0.03% (i.e., 0 transformants out of
3280 seed plated).

Controls:   0.48, 0.56%.

(These experiments have NOT been repeated)


Expt. 5:    5% Sucrose is very important:

Used 10 mM MgCl2 and Silwet L-77 instead of standard inoculation
medium: 0.033, 0.068% transformants.

Used just 5% sucrose and Silwet L-77 instead of std. inoculation
medium: 0.2, 0.81% transformants.

Replacing sucrose with 5% mannitol or 5% glucose gave no transformants
(note: need to repeat using equal osmolarities).

Controls: 0.48, 0.56% transformants.

(These experiments have NOT been repeated)


Expt. 6:     Vacuum is not required but gave us more transformants and
more consistent results.  Silwet L-77 helps when dipping but does not
seem very important if using a vacuum:

Suspended bacteria in standard inoculation medium and dipped plants
into this for about 5 sec with mild agitation: 0.54, 0.9, 0.19, 0.245,
0.16, 0.36% transformants.

Dipping into suspension without the Silwet L-77: 0.13, 0.03%
transformants.

Controls: with Silwet and vacuum: 0.87, 0.55, 0.99, 0.42, 0.82, 0.38%
transformants.

Controls: with vacuum but without Silwet: 0.84, 0.48% transformants

(Experiments have been repeated)


-Parenthetical note:  One can also apply Agro by spraying flowers until
they are drenched.  We, and Jeff Maughan in Lila Vodkin's lab here at
University of Illinois, have tried this and it gave a good rate of
transformants.  However, it is messy, the bacteria are sprayed all over
the place, and Silwet L-77 is not good for ingestion (or eye-contact -
contact lens users in particular should beware).



OUR CURRENT PROTOCOL:

1)      Grow plants and prune back bolts if necessary to encourage numerous
unopened buds at time of infiltration (we clip primary inflorescence at
its base, let secondary inflorescences grow until they start to show
open flowers and these bolts can also be clipped if Agro strains are
not ready).


2)      Grow Agro GV3101 in LB (5g salt/L) until late log/early stationary
phase. Pellet cells and resuspend to OD600 = 0.8 in 10mM MgCl2, 5%
sucrose, 0.005% Silwet L-77.  44nM BAP can also be added (optional).


3)      Inoculate by one of the two following methods:

     a)  Submerge in Agro suspension and draw vacuum until solution

   bubbles vigorously, then quickly release the vacuum, or:

     b)  Dip and gently agitate for approximately 5 seconds.


4)      Cover plants with plastic dome overnight.


5)      Grow plants until seeds are mature and dry.


6)      Sow bulk seed on selective medium to identify transformants. For
selective medium we use: 1/2 strength MS Medium (Sigma M-5519), 0.8%
Agar (Sigma A-1296), selectable antibiotic such as kanamycin at 50-60
ug/ml.


The keys to successful use of this protocol seem to be growth of
healthy plants, use of 5% sucrose, use of an Agrobacterium strain that
contains the desired binary plasmid (a number of people seem to be
fooled during Agro strain construction), and treatment of plants at a
time when they have many flower buds  forming.  We are as surprised as
anyone by the apparent unimportance of many other parameters, and
encourage others to explore the transformation procedure and publicize
their findings.





Steven J. Clough

Department of Crop Sciences

University of Illinois at Urbana-Champaign

1201 W. Gregory Dr.

Urbana, IL 61801



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