I want to select transgenic Arabidopsis seeds using BASTA on MS medium. My mutant does not survive well on the soil and want to initially select the T0 seeds on BASTA in MS medium. Can anyone tell me the appropriate concentration and brand of BASTA to be used for MS medium? Thanks!!
We use 10 ug/mL. Some people use 5 ug/mL and some use 15 ug/mL. Filter-sterilize the BASTA solution and add to cool-down medium.
Also, see RG post: https://www.researchgate.net/post/Which_concentration_of_Glufosinate-ammonium_should_I_use_for_selection_of_the_transformed_Arabidopsis
We use Glufosinate-ammonium (also known as phosphinothricin or BASTA) purchased from Sigma at a concentration of 10 ug/mL plus 150 ug/ml timentin (Ticarcillin disodium salt -also from Sigma) to inhibit growth of any Agrobacterium that may be trapped under the seed coat. I have tried with 5 ug/mL BASTA as well but apparently the strength is not higher enough for my selection.
We use PPT (phosphinotricin) from Duchefa (http://www.duchefa-biochemie.com/product/details/number/P0159), working concentration in MS for plants/seeds 50 µg.mL-1, for cell suspensions - 25 µg.mL-1
Hi, Teotia
You need to do a series of concentrations testing to find out the best selection concentration.
Regards
If your bar gene is well-expressed, say by 35S, then this is an excellent selection at 5-10 mg/L. Arabidopsis seedlings germinate and then bleach. 10 mg/L (i.e. 0.05 mM or 50 µM micromolar) is the final concentration to be used.
I recommend you to look for the specific information about the selection with the vector you are working.
Generally, we use 10-15 mg/L in either MS media or Hoagland's. Put out some wild-type seeds (with no BASTA resistance) and a transgenic line with BASTA resistance as controls to see if the concentration is appropriate for selecting transformed T0s.
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