Answers for Transient Agrobacterium transformation

Raimund Tenhaken tenhaken at rhrk.uni-kl.de
Sat Aug 28 13:15:59 EST 1999


As several people were interested in protocols for transient Agrobacterium
assays I now send some of the answers.
Thanks for all the kind comments

Raimund

The original question:

>We are looking for a protocol for transient transformation of Arabidopsis
>plants by Agrobacterium.
>In particular, which density of Agrobacteriua gives best results? Is it
>necessary to resuspend the Agrobacteria in MS-infiltration medium used for
>dip-transformation?
>Thanks for comments.
>
>Raimund
>

***************************************************************************

Hi Raimund,

I don't have an answer to your transient assay. However, you made a
reference to the 'MS-infiltration medium used for dip-tranformation' and it
is not necessary to use MS medium to transform by dipping. We have had very
good results in Andrew Bent's lab by simply resuspending in 5% sucrose and
0.05% Silwet. Andrew has the protocol on his web page:

http://www.cropsci.uiuc.edu/~a-bent/protocol.html

Regards,
Steve Clough

Steven J. Clough
Dept Crop Sciences
University of Illinois
388 ERML
1201 W. Gregory Drive
Urbana, IL 61801

Phone: 217-244-6150
FAX:   217-333-4582

*******************************************************************


Hi there,

In our lab we routinely use a Vacuum Infiltration based transient
expression experiment. Usually to test constructs during the course
of a stable transformation. We used it both for Arabidopsis and for
tobacco.
The protocol is basically as follows:

Immerse the desired amount of seedlings in appr. 5 ml of Agro
suspension (Suspension is 1/2 MS medium supplemented with 200 uM
Acetosyringone and 20 ul/100 ml Silwet L-77. OD600 has to be between 0,6
and 0,8, we noted that for
LBA4404 0,8 is too high, but for other strains it doesn't make a lot
of a difference). Mix it and apply a vacuum for 5 minutes.

Immediately wash the seedlings in liquid 1/2*MS medium and blot dry
on a piece of sterile Whatmann paper. Then transfer the seedlings
onto a 1/2*MS agar dish. Make sure each seedling has its root in full
contact with the medium.
Leave the cocultivation in the light at standard Arabidopsis growth
chamber conditions for three days.

Usually we get high frequencies of T-DNA transfer using this method.
A reference for the protocol that this one is adapted from is found
in Rossi et al (1993) PMB rep.11:220-229.

I hope to be of help and if you have any questions regarding the
protocol be welcome to contact me,

Dolf Weijers

----------------------------------------------------------------------------
------------------------------------------------
Dolf Weijers
Dept. Developmental Genetics
Institute of Molecular Plant Sciences
Leiden University / Clusius Lab
Wassenaarseweg 64, 2300 AL Leiden
The Netherlands
Telephone : 31-71-5274835
Fax       : 31-71-5274999
E-mail    : Weijers at rulbim.leidenuniv.nl
----------------------------------------------------------------------------
------------------------------------------------

*********************************************************************


Raimund,

This experiment has been done using a range of different densities and
other variables.  The reference is:
Rakousky, S., Kocabek, T., Vincenciova, R., and Ondrej, M. (1998).
Transient beta-glucoronidase activity after infiltration of Arabidopsis
thaliana by Agrobacterium tumefaciens.  Biologia Plantarum 40(1): 33-41

As for is the MS media important, look at a earlier post by Andrew Bent:

http://www.bio.net:80/hypermail/ARABIDOPSIS/9707/0015.html

He said
"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.

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."

Hope this helps.  Reply to: j.mylne at botany.uq.edu.au

Josh

--
Josh and Susan
74 Munro Street
Auchenflower 4066
Ph 3217 7402
E-mail: s324806 at student.uq.edu.au


******************************************************************




******************************
Raimund Tenhaken
Universität Kaiserslautern
Fachbereich Biologie, Geb. 22
D-67653 Kaiserslautern
Germany

Phone 49 631 205 3040
FAX   49 631 205 2600
E-Mail tenhaken at rhrk.uni-kl.de
******************************
---




More information about the Arab-gen mailing list