Mots-clés : Arabidopsis thaliana - ressources biologiques - mutants d’insertion - écotypes - populations de lignées recombinantes - collection - core collection

DNA isolation from Arabidopsis seeds

Arabidopsis culture

• seed sterilization :

- Dissolve 1 bleach pellet (Bayrochlore, Bayrol) in 40 ml H2O, add a few drops of Teepol or Tween (prepare fresh).
- Dilute the previous solution 1/10 in 95% Ethanol to make the sterilization solution (SS).
- Add 10 ml of SS to each tube of seeds, and incubate 5-7 min at room temperature with constant, gentle agitation.
- Rince twice with 10 ml 95% ethanol.
- Let the seeds sediment, and carefully remove as much ethanol as possible. (It's possible to invert the tubes, but be careful !).
- Leave the tubes open under a sterile hood overnight.

• in vitro culture medium:

We use Arabidopsis medium diluted 1/2 (AtM/2), without sucrose (Estelle & Sommerville, 1987, MGG 206 : 200).

Autoclave (120°C, 20 min)

Keep at -20°C.

• Sprinkle the surface-sterilized seeds on a 14 cm agar plate containing Arabidopsis culture medium (AtM/2), covered with a round filter paper (Whatman 3MM). Seal the plates with a gas-permeable surgical tape.

• Synchronize germination by a cold treatment at 4°C for 48 hours.

• Place in the growth chamber under the following conditions : photoperiod 16 h day (100-150 µE/m2/s) / 8 h night ; temperature 20°C day / 15°C night ; humidity 70%.

• After 10-15 days of culture, plantlets (2-leaf rosettes) are ready for DNA isolation. Each plate should yield 3-6 g fresh weight.

• Gently scrape the plantlets from the filter paper using a razor blade. The plantlets are weighted, and frozen in liquid nitrogen for future use.

DNA extraction

Reference : Doyle & Doyle, Focus 12 : 13-15

• Prepare an adequate amount of Extraction Buffer + ß-mercaptoethanol, and preheat at 60°C

• Weigh samples, and freeze them in liq. N2. Grind <2 / 3 / 5 g of fresh or frozen tissue to a fine powder, in liquid nitrogen with a mortar and pestle.

• Add liquid nitrogen, transfer directly to a 30 ml tube and wait untill all the nitrogen has evaporated. Add 7,5 / 11 / 20 ml preheated EB, mix well, and cap the tube. Incubate 30 min at 60°C with occasional swirling.

• Extract with 7,5 / 11 / 20 ml ml Chloroform : isoamylalcool (24:1), centrifuge (swinging bucket rotor : 5000 rpm, 10' ; other : 10000 rpm, 10'), and recover the aqueous phase.

• Add 6 / 8 / 14 ml isopropanol, and mix gently to precipitate the nucleic acids. Centrifuge (10000 rpm, 10') and decant the supernatant.

• Wash the pellet with 10-20 ml of ethanol 70%, centrifuge (10000 rpm, 5'), decant supernatant.

• Dissolve pellet in 500 µl TE, transfer into an Eppendorf tube. Add 50 µl sodium acetate 3 M, 500 µl isopropanol, centrifuge 5', decant, wash with 70% ethanol, dry on the bench.

• Dissolve DNA in 100 µl TE / g of fresh tissue extracted. Expect a concentration of 100-200 ng/µl. Store at -20°C.