Selection of BASTA resistant Arabidopsis transformants in soil.

 

* To make working stock, dilute Finale BASTA (120 mg/ml) 1:1,000 in water and add 500 ÁL of Silwet-77 (surfactant) per liter. Store diluted solution at 4 C. (BASTA is also known as phosphinothricin or glufosinate; trade names Finale, Ignite, or Liberty).

 
 ***Notes from internet:BASTA selection on Petri plates 


We use 50 mg/L of BASTA (glufosinate ammonium) obtained from Crescent
Chemical (1-800-877-3225) on agar plates with standard nutrients and 30
g/L glucose.  We place our plates in the refrigerator for 2-3 days after
plating.  This seems to help germination rates.
After removal from the refrigerator, plates are placed under regular
fluorescent (cool white) lights.
Hope this helps.
Dave Meinke (meinke at okstate.edu)
***
=46or selection on plates you'll need ppt (glufosinate-ammonium, PESTANAL),
which I obtained from FLUKA/ Riedel-de Haen. I used 10mg/l and this
selection works very good.
Best wishes,
Eric (Eric van der Graaff Egraaff at botinst.unizh.ch)
***
We usually do it on soil but it is possible on agar plates as well:
	BASTA Selection on soil
1) Plant seeds at high density on regular soil mix (whatever you use
otherwise to make A. th. happy).
2) Stratify for 4 days if seeds are freshly harvested and dried.
3) When you put them to the green house spray trays with 500 ml /
square meter of a diluted commercial Basta/ Liberty solution at 0.25 g/ l.
Use the regular Basta from Hoechst (yellow bottles, which contains 200 g/ l
glufosinate-ammonium, isomer mixture); in the US get Liberty which,
however, may contain another concentration of glufosinate.
4) Repeat spraying 2-3 times every 8-10 days until most of the germinated
seedlings are dying (yellowing of cotyledons, drying) and a few plants stay
green.
5) Harvest seeds from surviving primary transformants.
6) Segegration analysis of resistance can be done in the same way if you
plant seeds on soil and count the number of surviving germinated seedlings.
	"Basta" selection on agar plates:
use 25 ug/ml phosphinotricin (pure, not the isomer mixture used in the
commercial formulation). You may add it before autoclaving because it is
rather heat stabile. I was told (and therefore never did that) that it is
not wise to use the formulated stuff when autoclaving because it would
start foaming (the info was from Hoechst people). However you might try to
add it after autoclaving at a concentration corresponing to 25 ug/ml, i.e.
up to 50 ug/ ml Basta.
Good luck,
Tony Schaeffner (schaeffner at gsf.de)
***
I use 100mg/l for spray on the agar plate and I usually spray several
times to see the effect. Hope this will answer your question.  Good luck,
Li Zhou (zhou at genetics.mgh.harvard.edu)
***
We use the concentration of 30 mg/L of BASTA for the seedlings in the
soil.  For the seedlings on the agar media, we add BASTA while we are
preparing the agar media (the concentration of 3 mg/L). Both
concentrations work well.
=8A regarding BASTA selection on the plates.
=46or me, it was not that successful. Since I had to spray ABA on Arabidopsi=
s
seedlings on the plates (which was the way we find our mutant phenotype)
before BASTA selection, that caused the contamination that made it difficult
for me to tell the difference between BASTA sensitive ones and the
fungi/bacteria infected ones.
Hope this helps.
Byeong-ha ("Byeong-ha Lee" byeongha at U.Arizona.Edu)
***
You can select Arabidopsis for BASTA resistance on Petri plates by using
PPT(Phosphinotricine, Duchefa) at a final concentration of 5 ug/ml in MS
medium. It works for selection of different ecotypes such as Col, Nd-1 or
WS ... PPT is soluble in water. I prepare a stock solution at 10 mg/ml and
I sterilize it by filtration on 0.22 uM. You can stock this solution at
-20=B0C for a few months. Petri plates with PPT can be stored for two weeks
at 4=B0C.
Don't hesitate if you have more questions.
Laurent DESLANDES (phD student) (ldesland at toulouse.inra.fr)
***
Sowing seeds directly on basta containing medium results in a lot of
escapes, presumably simply because as soon as the seed germinates,
the cotyledons are not in direct contact with the medium and
herbicide.
However if you sow the seeds in top-agar containing Basta, you can
easily circumvent this problem and even have efficient selection at
high density.
Sincerely,
David Bouchez (bouchez at versailles.inra.fr)
***
Can be done and its fairly routine, cant remember what the dose is, it is
lower than the dose is for spraying though.
Regards
MikeSalter (mgs5 at leicester.ac.uk)
***
Hi Anna, that is exactly what we're doing here in the Benning lab. I have
found three things to be critical, the first is that the plates have no
Agrobacterium or fungus on them, if they do the agro and/or fungus will
break down the BASTA and you will select artifacts. The Dellapena lab has
recommended Clavamoxin and I have found that it works perfectly in getting
rid of Agrobacterium (clavamoxin can be obtained from your local vet, just
tell them what it's for and they'll set you up). The next is that the
plants are extremely sensitive to BASTA, even the resistant ones, and I
found that 0.5X was the best level (25 uM), added when the agar has cooled
to just above the geling point. The third is that even though they are
resistant, left too long on the plates the plants will begin suffer from
the BASTA. I think it's best to pick off the resistant ones and then
rescore the seedlings by spraying or sowing the progeny on plates again.
This is all based on our experiences with pSKI015. Good luck
Alex(cernac at pilot.msu.edu)
***
I am working on this very same thing right now.  I think using
phosphinothricin (from Sigma) or glufosinate (from Riedel-de Haen) at the
rate of about 7.5mg/L works.  I've used 5mg/L and 10mg/L recently.  5mg/L
was a little too low and 10mg/L was a little too high.  But remember, given
the way the herbicide works, the concentration that works successfully can
be dependent upon the intensity of the light that the plants are grown
under.  You may want to try plating seeds on different concentrations(in
the range of 5 to 10mg/L) to find what works well under your current growth
conditions.
Good Luck,
Colleen McMichael (Colleen.McMichael at usa.dupont.com)
***
It worked fine for me on GM (or any other germination media) with 10
microgr/ml of ppt.
Regards,
Victor Klimyuk (klimyuk at icongenetics.de)
***
I have used basta selection on plates with success. I used a
concentration of 10 microg / ml of PPT (non-trade name for basta - I have
purchase details for UK only) in usual B5 or MS media (treated basta as an
antibiotic). Then plated surface sterilised seeds on plates as usual and
incubated as usual. Resistant seedlings (ie those which didn't die!) were
obvious after a week or so.
Hope this helps,
Vicky Larner (vicky.larner at bbsrc.ac.uk)
***
I tried it a couple of times using 5mg/L PPT (the active ingredient of
BASTA), and it worked quite well. Someone found it slightly leaky (compared
with Kan selection). Proper concentration of PPT might depend on sources of
the chemical, but you can try it at around 3-10mg/L.
Yiji Xia (xia at akkadix.com)
***
If you are just doing segregation analysis or want BastaR plants for
immediate harvest or analysis, you can do it in non-sterile petrie
dishes containing 0.0066% Basta.  The plants will not grow past the
cotyledon stage but it is big enough to see Basta resistance vs
non-resistance.
Josh Mylne (s324806 at student.uq.edu.au)
***
Here are my notes for titration of BASTA (MW195) on MS-sucrose plates
with BASTA sensitive and resistant plants: 7d (after planting)
observations; 0-12.5 uM, plants identical; 25-50 uM, 2 versus 4 lvs,
chlorotic vs green lvs, roots 1 vs 2cm.  100 uM, 2 versus 2 lvs,
chlorotic vs green lvs, roots =BD cm both.  Petri
plates work, but it does sort of defeat one of the advantages of
phosphoinothricin which is the ability to apply it as a pre-emergent or
post-emergent herbicide in a soil environment without seed
sterilization.  I do have a protocol for that if you are interested
Cheers, Jim Tokuhisa (tokuhisa at ice.mpg.de)
****
****
****

Plus some info from the ARABIDOPSIS/bionet.genome.arabidopsis Newsgroup
Archive:

The overall advice is that this form of selection works,
and resistant seedlings can be distinguished about one week after
germination. About 50 =B5M phosphinothricin (PPT, the active ingredient in
Basta) is sufficient for the selection
***
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).
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.
***
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.
***
I used 5-10 ug/ml PPT (estimated, BASTA =3D ~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.