So I have always used Hygromycin/Kanamycin selection for Arabidopsis but this is my first time I am using methotrexate for selection of T0 transformed plants. Plants with hygromycin resistance I generally keep in dark and in3-4 days the one with resistance grows well.
I found only 2-3 papers where methotrexate has been used and it was not clearly mentioned that if there are more conditions for methotrexate selection other than just ading methotrexate to MS medium.
The working concentration I am using is 0.1mg/L.
Please let me know if you have experience with Methotrexate selection.
Thanks in advance.
Hi there, I've used MTX regularly and have created several mutagenic Arabidopsis lines using this selection. It's very effective and easy to use. Just place it in the medium at a final concentration of 100 ng / mL* and sterilise / plate your seeds from transformed plants as you normally would.
MTX affects root growth by inhibiting the function of Dihydrofolate reductase, part of folate metabolism pathway, and the resistance gene is a variant form of the DHFR gene that is resistant. So sensitive seedlings will germinate and will start to grow a shoot and root, but will stop when the root is ~1 mm long. Resistant individuals will produce a healthy full-length root - quite easy to find in the plate.
* this is 0.1 mg / L as you say: to get to this low concentration, I dissolve the MTX powder in DMSO to a final concentration of 10 mg / mL (should be strongly yellow), then make a working stock by a 1:100 dilution to 100 ug / mL (weakly yellow). Then use this working stock at a 1:1000 dilution in the media (so 50 uL in 50 mL media) to final concentration of 100 ng / mL. Make the stocks fresh, it doesn't seem to work so well when it's been frozen / thawed. Good luck!
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