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Basta/PPT selection on MS agar

Scott Bowling sbowling at acpub.duke.edu
Wed May 18 08:26:17 EST 1994


Arabinetters,

Thanks to all of those who responded to my inquiry about Basta selection on
MS agar plates.  The overall advice is that this form of selection works,
and resistant seedlings can be distinguished by about one week after
germination. About 50 µM phosphinothricin (PPT, the active ingredient in
Basta) is sufficient for the selection.  Following are the responces I
received.
-Scott

Thomas Altmann, IGF Berlin:
 altmann at MPIMG-Berlin-Dahlem.MPG.DE
I used pure phosphinotricin for selection of BASTA resistant Arabidopsis 
seedlings harboring a p35S-bar gene. This worked nicely at a concentration 
of 0.05 mM. The sensitive seedlings stayed small and white while the 
resistant ones grew well; they could be distinguished already about one
week 
after plating (on half concentrated MS-medium with 1 % sucrose).

Brian Osborne:
 bosborne at nature.Berkeley.EDU
If your bar gene is well-expressed, say by 35S, then this is
an excellent selection at 5-10 mg/l. Seedlings germinate and
then bleach.

David A. Patton, Ciba:
 PATTOND at a1.abru.cg.com
The active component of BASTA is glufosinate-ammonium, also known as 
phosphinothricin (PPT).  I have had good success with glufosinate selection
on 
germinating Arabidopsis in 1/2 MS + 1% Sucrose + 50 uM (micromolar)
glufosinate 
+ 0.7% agar.  I routinely spread 5,000-10,000 seeds onto large 150mm plates
for 
selections where only a few transformants are expected (ie in planta or
vacuum 
infiltration) and few hundred/ plate if doing segregation ratios.

You can get 250 mg of technical grade glufosinate-ammonium from Crescent 
Chemical Co. (Hauppauge, NY) for $85.00.  The stuff is stable as rock in
water 
at 50 mM (1000X) when stored at 4 C.  The MW is 195, so 250 mg should be
enough 
for at least 25 liters of media.  

Mary Honma, Plant Gene Expression Center:
 honma at garnet.berkeley.edu 
I used 5-10 ug/ml PPT (estimated, BASTA = ~20 % PPT in detergent) in GM
agar for selection.  The leaves looked reasonably healthy, but the roots
barely elongated.  More recently I have used purified PPT and the plants
look fine.  Purified PPT is available as GLUFOSINATE AMMONIUM (#45520.01)
from Crescent Chemical Co., 1324 Motor Pkwy, Hauppage, NY 11788 Tel:
516-348-0913.  Hope this helps.



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