Questions about Arabidopsis suspension cultured cell transformation

[Hongwei Zhao <zhaoh@MUOHIO.EDU>]

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)