ARABIDOPSIS CELL SUSPENSION AND ROOT LIQUID CULTURE
 
Last Update: December 2006
 
Reference: Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B (1992) A protocol for transient expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiol Biochem 30 (1): 123-8
 
PREPARE SOLUTIONS
1. Cell suspension media (CM) (1 L): Mix 800 mL of dH2O, 3.2 g of Gamborgs with minimal organics (Sigma G5893), 20 g of sucrose, 0.5 g of Mes, 2.2 mL of 0.5 mg/mL 2,4D pH to 5.7 w/ 1 M KOH, and dH2O to 1 Liter (Autoclave)
2. 0.5 mg/mL 2,4D (50 mL): Mix 48.5 mL of dH2O, 25 mg of 2,4D, 1.5 mL of 1M KOH, Shake into solution (Filter (22mm) and store at 4oC)
3. Plates: Add 6-7 g of Phytoagar per liter of media
4. Seed germinatin media (SG) (1 L): Mix 800 mL of dH2O, 10 g of sucrose, 4.3 g of MS salts, 0.5 g of Mes, 1 mL of 1000X vitamin stock pH to 5.7 w/ 1M KOH, and dH2O to 1 Liter (Autoclave)
5. 4M LiCl (100 mL): Mix 17 g of LiCl with 100 mL of dH2O (Autoclave)
6. 1000X vitamin stock (100 mL): Mix 10 g of Myo-inositol, 100 mg of Thiamine, 100 mg Pyridoxine, 100 mg Nicotinic acid, dH2O to 100 mL, Store at 4oC (Heat in ~60oC water bath if precipitation occurs)
   
PROCEDURE - CELL CULTURE
1. Suspension cultures are made weekly by subculturing 8-10 mLs of a week old "mother culture" into 50-55 mls of fresh CM in a 250 mL flask
2. Cultures are grown in the dark or light at room temperature (22-25oC) on a rotary shaker (120 rpms) for a period of one week
3. Longer or shorter periods of growth are possible as well as different sized cultures (40 mL to 1 L cultures have been used successfully)

NOTE: The idea is to dilute the week old culture 1:5 with fresh media. The length of the growth period varies, with 60 mL cultures taking nearly a week to reach stationary phase while 1 L cultures take at least 2 to 3 weeks to reach a similar stage. Also, spinning down the week old culture or draining the old media by passing the culture through a 80 mm mesh would help in getting rid of the old media. It seems that under certain growth conditions old cultures tend to secrete substances that, when transferred to a new flask, will inhibit the proper growth of the culture. Lack of growth, light green to yellowinsh cells, and non-clear (clowdy) media after a single week's growth of a 60 mL culture are some symptoms found when old media is transferred along with the cells to a new flask.
   
PROCEDURE - ROOT LIQUID CULTURE
1. Sterilize Arabidopsis seeds
1.a. Place seeds in an appropiate tube (15 mL Sarstedt tube) and add ~10 mL of a 80% Ethanol, 0.1% Triton X-100 solution
1.b. Rock seeds for 10 minutes (use a rocker, invert the tubes; anything that will keep the seeds from settling to the bottom of the tube)
1.c. Let the seeds settle to the bottom of the tube and draw the solution out (be careful not to take the seeds too)
1.d. Add ~10 mL of a 30% Bleach, 0.1%Triron X-100 solution and again rock for 10 minutes
1.e. Let the seeds settle to the bottom of the tube and draw the solution out
1.f. Add ~10 mL of sterile dH2O and again rock for >10 minutes
1.g. Repeat these washes at least two more times
2. Seeds are sterile. Seeds may be store in water for days/weeks at 4oC (3-5 days at 4oC [cold treatment] helps in getting uniform germination)
Optional: Sterilize with 50% bleach/0.02% Trition x-100 for 7 mins (or a similar treatment using 70% alcohol/triton), drain the tubes with seeds of most of the liquid, squirt in 95% alcohol and immediately dump the contents on a clean piece of filter paper. After alcohol evaporates, transfer seeds from the filter onto agar. The whole procedure is done in a sterile environment
NOTE: if possible germinate the seed in Seed germinatin media (SG) plates prior to starting the culture
3. Add 3-5 seeds (or 1-3 seedlings) to a 500 mL flask containing 250 mL Seed germinatin media (SG)
4. Grow under continuous light on a rotary shaker (120 rpms)
NOTE: under these conditions plants can be "harvested" in as little as two weeks
NOTE: plants should not be left for longer than a month (overgrowth and depletion of nutrients)