gentamycin selection: summary and thanks

Yulan Cheng yulanc at waksman.rutgers.edu
Tue Aug 21 10:55:20 EST 2001


Hi,

I asked for help from this news group about the selection of
transformants on gent plates.  I got a lot of very helpful suggestion.
According to these, I tried different condition: different gelling
agent, different media type and different gent concentration,  and
finally  I got perfect selection.  Here, I want to thank them all for
their kindly help.

To summarize, the gelling agent is very critical for the efficiency of
gent on the plates. I did observe that gent got precipitated in the
media when i used our normal agar. However, on this kind of plates, 150
ug/ml gent still worked.  If use purified agar, the concentration can
drop down to probable 60 ug/ml.

The type of medium and the strengthen of salts in the medium also affect
the selection to some degree.  1/2 MS looks to work  a little better
than 1X MS.

Seeds can not be plated too dense.


I also chose some email i got from those people that helped and paste
below.



Hi Yulan,

I have used the pPZP121 vector and gentamycin selection very
successfully at the same concentration as what you are using (100
ug/ml), but I used regular floral dipping, not vacuum infiltration.
Anyway, you may be interested in those few things:

- The plants DO develop to a certain extent. Cotyledons will form on
almost all the plants, but development (appearance of true leaves and
development of roots) will proceed only for the transformed plants. It
takes 10-14 days to see that clearly. The non-transformed plants will
bleach over that period.

- What type of gelling agent do you use? I have noticed that, in my
hands, Phytagel (Sigma) should not be used with gentamycin. The
antibiotic precipitates, so I guess it becomes inaccessible to the
plant's roots (but that is definitely just a guess).

Good luck!

Francois


Yulan: Maybe you are expecting the non-transformed ones to become yellow
or
white as with kanamycin? Gentamycin is not such a strong antibiotic.
Make
sure you don't plate them too densely (1,000 per plate max), then let
them
germinate for two weeks. Then turn over the plate and look for seedlings

with LONG roots. Grow some nontransformed plants on Gen and some control

plants without Gen if you're not sure what to expect. 100mg/l Gen is
enough. - Albrecht.


Use 1/2 strength MS salts and 1% sucrose. I think this will solve ur
problem


Hi,  I am replying to your message on the arabidopsis newsgroup.  I have

had the same problem using 100 gentamycin.  What I have done is look at
the roots.  The resistant plants have strong roots that hit the bottom
of the plate and grow along the bottom.  Sensitive plants do not have
roots that grow into the agar or only grow in a small amount.  However,
it is a pain to find the respective seedlings with the good root
growth.  i would be interested to hear if you find an easier solution to

this problem.  Thanks  Chris


I think you should not plate the seeds too dense. That is critical some
time.


Hi  Yulan
I used the pPZP vectors too, somewhat unfortunately.
As far as transformation, you will see a difference later
in growth, the cotyledons usually are green in the beginning.
I found that 100ug/ml to be too much for my plants, this may be
ecotype dependent, you may want to play with different concentrations.
I must warn you that gentamycin selection is not as easy or nice as
Kan, but it is doable, you will just want to determine you best
concentration
and be patient.
Good luck.
Nadi


Dear Yulan,

I have used the following conditions to select for Arabidopsis seedlings

transformed with pPZP221:

Put out surface sterilized seeds onto MS agar containing 3% sucrose with
200
ug/ml carbecillin and 75 ug/ml of active Gentamycin (consult supplier
for
actual potency, we typically get 65 to 75% effective gentamycin);
stratify
the seeds for 72 hrs at 4C in the dark; transfer plates to 12 hr Light,
12
hr dark, 100 uEi cool white light, 22C; by day 7 all seedlings are still

green and have expanded cotyledons; by day 10 only transgenic seedlings
have
true leaves and their cotyledons are as fully expanded as similar
seedlings
grown without gentamycin, sensitive seedlings will never grow further
and
their cotyledons do not fully expand while truly resistant seedlings
will
continue to grow normally in the presence of the gentamycin.  The gent
selection is not as obvious as Kan or herbicides on soil but it can
still
work.

sincerely,

joel


Yulan,

We use pPZP222 for gent resistant transgenic plants. Our selection is 60

ug/ml. However, the agar you use as a gelling agent will dramatically
change
the efficacy of the gent for killing sensitive, non-transgenic plants.
We
use phytagar for selection media. For non-selection we routinely use
phytagel since it gives clearer phenotypes for photomorphogenic mutants
(ie
phyB). People in my lab have mixed up the two agars and found that 60
ug/ml
doesn't work with phytagel. The same is true for kan. If you suspect
this is
the case, then doing a kill curve with wildtype plants should sort this
out.

Other possibilities for nice green seedlings could be that the media was
too
hot when the gent was added (or that the gent was added before
autoclaving).
Along that line of thought, it is also possible that the gent is bad due
to
improper storage or manufacturing. In general, gentamycin is a little
trickier to use than kanamycin. The window where resistant plant live
(and
are healthy) where as sensitive plants die (ie are clearly dead) is
fairly
narrow. If your seeds are planted too densely this will also cause a
problem
as the antibiotic gets degraded or taken up by some plants and then the
others survive the selection.

Sincerely,

Michael



---




More information about the Arab-gen mailing list