Transformation by vacuum infiltration

Steven J. Clough sjclough at uiuc.edu
Thu Jul 3 20:59:21 EST 1997


<x-rich><fontfamily><param>Helvetica</param><bigger><bigger>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.
</bigger></bigger></fontfamily><bigger><bigger><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>A few notes concerning
the poster and this message:
</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>-- 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.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>-- We decided a few
days before the meeting to present the poster, hence there was no
abstract in the meeting
book.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>-- 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:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>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.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>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.</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>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.
</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Best
wishes,</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Steve Clough and
Andrew Bent</fontfamily><fontfamily><param>Times</param>




</fontfamily><fontfamily><param>Helvetica</param>___________________________
_</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Summary of
Poster</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Poster Title:
Transformation by vacuum infiltration with Agrobacterium--the vacuum
(and other factors) are not necessary.
</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Goals of the
project:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>   -Simplify / improve
infiltration protocol.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>   -Determine which
components are essential for successful
transformation.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>   -Use what we learn
to attempt similar transformation of other plant
species.</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Standard inoculation
medium that we use in our
lab:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>       1/2 strength MS
medium with vitamins (we use Sigma
M-5519)</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>       5%
sucrose</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>       44 nM
Benzylaminopurine (BAP)</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>       0.005%  Silwet L-77
(can be purchased from Lehle seed at
http://www.arabidopsis.com)</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>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.  </fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>More detailed notes on
our older "standard procedure" can be found
at</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>http://www.bio.net:80/hyper
mail/ARABIDOPSIS/9312/0083.html</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>A number of related
protocols with subtle modifications and comments can be located by
scanning the BioSci news group archives of the Arabidopsis news group.
This information is not essential to your effort, but if you are
interested you can look on the WWW under:
</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>http://genome-www.stanford.
edu/cgi-bin/biosci_arabidopsis.
 See also
http://www.bch.msu.edu/pamgreen/green.htm.</fontfamily><fontfamily><param>Ti
mes</param>


</fontfamily><fontfamily><param>Helvetica</param>All transformation
rates are expressed as Percent Antibiotic-Resistant Seedlings harvested
from originally treated
plant:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>  (#Km-resistant
seedlings/Total # seed plated) x
(100)</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>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. </fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>We are in the process
of further testing and the following data should be regarded as
"preliminary results" or "work in
progress."</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Expt. 1:    Effect of
vacuum on A. tumefaciens strain
EHA105.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>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.</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Expt. 2:
Pre-inducing Agro for vir gene expression did not seem to be
important:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>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%.
</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Also tried growing
cells in liquid LB cultures and then adding 100 uM Acetosyringone to
inoculation medium: 0.5, 0.4, 0.27% transformants.
</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Controls (std.
protocol): 0.82, 0.36% transformation.
</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>(This expt. has NOT
been repeated, note also that pre-induction was on plates and not
liquid).</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Expt. 3:     MS medium
does not seem to be
important:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Replaced MS medium
with 10 mM MgCl2: 0.5, 0.55, 1.13, 0.25%
transformants.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Controls: 0.48, 0.56,
0.82, 0.38%
transformants.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>(This experiment has
been repeated).</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Expt. 4:     BAP does
not seem to be very important, but it might have a slight effect.  Too
much BAP appears to be
deleterious:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Standard inoculation
medium minus the BAP: 0.29, 0.133%
transformants.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>44 uM BAP instead of
44 nM: 0.03, <<0.03% (i.e., 0 transformants out of 3280 seed
plated).</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Controls:   0.48,
0.56%.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>(These experiments
have NOT been repeated)</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>Expt. 5:    5% Sucrose
is very important:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Used just 10 mM MgCl2
and Silwet L-77 instead of standard inoculation medium: 0.033, 0.068%
transformants.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Used just 5% sucrose
and Silwet L-77 instead of std. inoculation medium: 0.2, 0.81%
transformants.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Replacing sucrose with
5% mannitol or 5% glucose gave no transformants (note: need to repeat
using equal osmolarities).
</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Controls: 0.48, 0.56%
transformants.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>(These experiments
have NOT been repeated)</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>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:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>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.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Dipping into
suspension without the Silwet L-77: 0.13, 0.03%
transformants.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Controls: with Silwet
and vacuum: 0.87, 0.55, 0.99, 0.42, 0.82, 0.38%
transformants.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Controls: with vacuum
but without Silwet: 0.84, 0.48%
transformants</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>(Experiments have been
repeated)</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>-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).</fontfamily><fontfamily><param>Times</param>



</fontfamily><fontfamily><param>Helvetica</param>OUR CURRENT
PROTOCOL:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>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 ;
these bolts can also be clipped if Agro strains are not
ready).</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>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).</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>3)     Inoculate by one of
the two following
methods:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>     a)  Submerge in
Agro suspension and draw vacuum until solution
bubbles</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>         vigorously,
then quickly release the vacuum,
or:</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>     b)  Dip and
gently agitate for approximately 5
seconds.</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>4)     Cover plants with
plastic dome overnight.</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>5)     Grow plants until
seeds are mature and dry.</fontfamily><fontfamily><param>Times</param>


</fontfamily><fontfamily><param>Helvetica</param>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</fontfamily><fontfamily><param>Times</param>.


</fontfamily><fontfamily><param>Helvetica</param>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
flowers 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.
</fontfamily><fontfamily><param>Times</param>





</fontfamily><fontfamily><param>Helvetica</param>Steve
Clough</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Department of Crop
Sciences</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>University of Illinois
at Urbana-Champaign</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>1201 W. Gregory
Dr.</fontfamily><fontfamily><param>Times</param>

</fontfamily><fontfamily><param>Helvetica</param>Urbana, IL
61801</fontfamily><fontfamily><param>Times</param>


</fontfamily></bigger></bigger>


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