Re: Questions about Arabidopsis suspension cultured cell transformation

[Michael Sullivan <mlsulliv@WISC.EDU>]

Are you basing your protocol on a published procedure? If so, are you
doing everything the same as in the published protocol? If not, you
might check out the paper by Forreiter et al. 1997 Plant Cell 9:2171,
whose protocol is based on that of An et al. for transformation of
BY-2 tobacco cells (which I've done a lot). There are likely other
published protocols as well.

A few things that might be problems with your protocol are:

You may want to do the transformation when the cell culture is
actively growing-- perhaps 3-4 days after subculturing and not the
day of subculturing. This has been shown to be important in some
systems (and Forreiter reported this was important).

Are you using an appropriate strain of Agrobacterium? Not every
strain works well for every plant or tissue.

Your washing/counterselection scheme seems unusual to me. Why do you
need to rewash after 6 and 7 days? If your counterselection is really
working, it seems like this wouldn't be necessary. You might try a
different counterselective antibiotic-- Timentin at 250 mg/L seems to
be commonly used.

Most protocols call for selection immediately after the co-
cultivation step. I don't see any reason to have a "grow out" period
as in your protocol. I would probably just plate the cells out onto
selective media right after the co-cultivation and washes. By growing
in liquid culture for so long, it will also be hard to know whether
any calli you get are independent events.

Do you know your untransformed cells will grow on solid media when
there is no selection?

Is your solid medium really gelled with Agar, or are you using a
gelling agent specifically for tissue culture?

I hope these suggestions are helpful.

Mike


On Oct 5, 2005, at 12:07 PM, Hongwei Zhao wrote:

> Hi, TCers
> Could anybody there give me some suggestions about the encountered
> transformation problems of my suspension cultured Arabidopsis cells? I
> listed my procedures and problems below, which is a bit long and tedious.
> If you happen to have experience about suspension cultured cell
> transformation, PLEASE take some time to read it and help me!
> Thanks in advance!
> I maintain my cell line (landsberg) in Skoog suspension cell culture which
> containing:
> MS (4.5g/L), VB5 (.112g/L), Sucrose (30g/L), MES (.59g/L), NAA (.5mg/L),
> and BAP (.05mg/L) by subdividing weekly.
> I transformed cell by Agrobacteria.
> The day I subdivide, I transform the freshly devided cells by
> co-incubating with agrobacteria containing constructs.
> Agrobacteria has been grown to OD600=0.4, and has been activated by 150uM
> of Acetosyringone for 2 hours.
> The co-incubation was kept 48 hours, then washed with MS media after
> spinning down, and added 100ug/ml of Mefoxine to stop agrobacterial
> propagation. And shake.
> Washing is repeated at the 6th and 7th days, respectively, and I always
> keep 100ug/ml Mefoxin in media.
> From the 7th day, I add selective antibiotics to media and I believe any
> cell survived will be the transformed. At the same time I transferred
> about 1 ml of culture on to solid MS which is identical to the liquid
> except 0.8% of agar.
> As the results, I never got callus directly from the selection on plates,
> instead, a few flasks keeping green have been observed. The selection by
> Kanamycin is better, while the selection by Glufoxinate (5ug/ml) against
> Bar resistance never works.
>
> Anybody can help me about it? Any help will be appreciated!
>
> Regards,
>
> Hongwei
>
> --
> Hongwei Zhao
> Department of Botany
> Miami University
> Oxford, OH 45056
> 513-529-4259
> 513-529-4243 (Fax)

---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)