Arabidopsis root culture

ARABIDOPSIS ROOT CULTURE

Last Update: December 2006

PREPARE SOLUTIONS

1. 1000X Vitamin culture mix (100 mL):

Mix 10 g of Myo-inositol, 100 mg of Nicotinic acid, 100 mg of Pyrodoxine-HCl, 100 mg of Thiamine-HCl, and dH₂O to 100 mL. Store at 4^oC. Heat in 65^oC water bath if precipitation occurs

2. Seed germination plates:

Mix 1 L of Root culture media and 5 - 7 g of Phytoagar (Autoclave). Pour plates. If antibiotics are to be added, cool media to <60^oC prior to antibody addition. Store at 4^oC

3. Root Culture media (1 L):

Mix 10 g of Sucrose, 4.3 g of MS Salts, 0.5 g of Mes, 1 mL of 1000X vitamin culture mix, and 800 mL of dH₂O. pH to 5.7 w/ 1M KOH and add dH₂O to 1 L (Autoclave)

PROCEDURE

1. Sterilize seed:

1.1 Wash seeds for 10 mins in a 80% ethanol + 0.1% Triton X-100 solution

1.2 Wash seeds for 10 mins in a 30% bleach + 0.1% Triton X-100 solution

1.3 Wash >3X for >10 mins with dH₂O

1.4 Add a sterile 0.7% agarose solution

2. Plate seed on seed germination plates

3. Pick individual seedlings (after >3 days) and place them in root culture media (previously aliquoted to appropiate sized vials)

4. Incubate rocking (250 rpms) for <1 month

5. Roots can be harvest and used for downstream applications