Jasmonic acid (JA) sensitivity assay

JA sensitivity root assay protocol (using agar plates with shelves)
Neva Laurie-Berry
Kunkel Lab, Washington University
October 2007

Make up plant growth media asfollows:

½ strength MS
1% (10g/L)sucrose
1% (10g/L) agar
pH 6.0
Methyl jasmonate (MeJA; at appropriate concentration)*
 
*MeJA must be added after autoclaving and letting agar cool to ~55oC.
In a typical dose-response assay we use: 0, 10 and 50 µM MeJA.
 
We use square Petri dishes, 100 x 100 x 15 mm, with grid. The grid allows easier
estimation and comparison ofroot lengths.
 
Pour dishes about halffull of media. It is best to let the platessit overnight to dry a bit.
 
Using a sterilized scalpel, cut a strip of media out of each plate. If using gridded plates, it
is easiest to cut out the top grid square. Thisforms a shelf of agarthat you will place your
seeds on. A diagram illustrating thisis provided below.
 
Use a toothpick to individually place sterilized seeds along thisshelf, centered with 
respect to the depth of the media. For plates of the indicated dimension, evenly space 15-
20 seeds along each plate.
Wrap the edges ofthe plate with Parafilm. It is essential that you use Parafilm or a similar substance that is permeable to O2 and CO2, but not to waterto prevent the platesfromdrying and shrinking asthis will allow the rootsto escape from the media at the thin spots, and begin to grow along the surface of the agar.
 
Plates should be placed vertically with the shelf asthe top side. This will cause the majority ofrootsto grow down into the media and remain within it(and thus exposed to MeJA) for the entirety ofthe growth process.


After the desired growth period (e.g. 10 days.), the roots can be photographed, measured, or otherwise examined.

 

When transplanting seedlingsfrom this condition, it is easiest to simply transplant a small agar plug around the rootrather than trying to extract the rootfrom the agar.