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Sterilization and Plating of Agrobacterium-transformed Arabidopsis Seeds

from the Sun Lab

Soak 100 µL seeds in water for 30 min.

Sterilize seeds in 0.5% SDS (make 2.5% stock: 2.5g/100 ml H2O, autoclave), 10% bleach for 15 min. ¿ (sterilization solution: in 10 mL: 1 ml bleach, 2 ml 2.5% SDS, 7 ml H2O)

Rinse 3x and then cold stratify for 2 days in 1 mL of H20 in dark.

Remove H2O and resuspend seeds in 0.1% sterile agar (0.1g agar/100ml H2O; autoclave) and plate one tube/plate. You may need to add an extra 1 or 2 mL of 0.1% agar to the plate to facilitate seed distribution. Let dry in flow hood

MS plates (full strength MS, 2% sucrose, 50ug/ml Hyg for plant selection (add 500ug/mL Carb or 200 ug/mL Cefotaxime to kill residual Agro when selecting T1 seed)

  • per 500 ml plates: 2.2g MS, 10g sucrose, 7.5 g agar
  • for kan or hyg (50ug/ml) = 25 mg (or 500 ul kan)
  • 500ug/ml carbenicillin = 250mg

Put in growth chamber under lights for 1 day (2 lights/bench for plates)

For hygromycin selection: wrap plates in foil for 3-4 days. Only transformants will elongate the hypocotyl.

  • If selecting on Kanamycin, no need to do this - seedlings will emerge, then non-transformed seedlings will die within ~2 weeks.

Return to full light until the cotyledons turn green and transplant to soil.

Note: once you have a transgenic line and are testing for segregation, 25 µg/mL Hyg is enough.

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