SEED STERILIZATION
1. Sterilize Arabidopsis seeds for 10 min in a solution of 50% bleach plus 0.1% SDS.
2. Rinse 5 times with sterile dH2O.
3. Place 50 seeds onto B5 medium plate (containing 50 ug/ml kanamycin or 10 ug/ml
phosphinothricin, whichever is appropriate). Also put into the medium 100 ug/ml timentin to inhibit growth of any Agrobacterium that may be trapped under the seed coat.
4. Place plates at 4�C for 2 days.
ROOT CULTURE
5. Germinate seeds in a growth chamber (23�C, 10 hr light, 14 hr dark) for 7-10 days.
6. Transfer seedling into baby food jar containing B5 medium plus timentin and grow for at least 10 days. Plants are ready for processing when the roots are long enough to get a minimum of 60 segments. Plants should be processed before a flower bolt emerges.
AGROBACTERIUM INFECTION
1. Place Arabidopsis roots into a Petri dish, and pour some sterile H2O into it.
2. Cut the roots from the plants and replace the plants back into baby food jars. Cut the roots into 0.3-0.5 cm long segments.
3. Transfer bundles of root segments onto MS basal medium without antibiotics.
4. Place 2-3 drops of bacterial solution (grow Agrobacterium to a density of 109 cells/ml [Klett=100] in YEP medium containing the appropriate antibiotics, wash the cells once in 0.9% NaCl, then resuspend the pellet in 0.9% NaCl at a concentration of 108 cells/ml [Klett=10]) to cover the root bundles and leave it for 10 min.
Notes: Centrifugation of bacteria is done in a microfuge at top speed for 1 minute. For certain experiments, you may wish to resuspend the bacteria at a Klett of 100, or a Klett of 1 or even less. All root segments must be infected within 2 hours of being cut. Do not leave the segments for longer periods of time before infection.
6. Remove most of the bacterial solution, seal the Petri dishes with Parafilm, and co-culture the bacteria and root bundles for 40-50 hours in a growth chamber at 20oC.TRANSIENT GUS ACTIVITY7. 40-50 hours after co-cultivation, rinse the segments with d H2O containing Timentin (100 ug/ml) or scrape off infected root segments on the surface of the medium. Transfer roots onto different types of medium according to the assay you are going to perform. For primary screening for rat mutants, separate roots into small bundles (up to 5 root segments/bundle). For secondary screening and quantitation, separate into individual root segments; do not use root bundles. You must have a minimum of 60 root segments per plate.
8. Incubate the plates at 23oC, and score in 4-5 weeks.
9. When scoring for the phenotype of tumorigenesis, not only must you record the percentage of root segments that give tumors; you must also indicate the morphology of the tumor (small yellow, large yellow, small green, or large green). The percentage of each morphology class should also be recorded (you may wish to use a bar graph in which the bar is divided into 4 sections of different colors, each color representing a morphology class).
1. Transfer root bundles onto Callus Inducing Medium (CIM) containing 100 ug/ml of Timentin, seal the plates with double layers of parafilm, and place them in a growth chamber.
2. Take out the root segments 2-4 days later and stain them in X-gluc solution overnight at 37�C or grind the segments in a 1.5-ml eppendorf tube and measure the specific GUS activity by a flurometric method.
TUMORIGENESIS ASSAY
1. Transfer the roots onto MS basal medium with 100 ug/ml Timentin.
2. Seal the plates with double layers of parafilm and place them in a growth chamber for 4-5 weeks. Around 2 weeks after infection, you should be able to see small tumors appear.
TRANSFORMATION TO KANAMYCIN- OR PHOSPHINOTHRICIN-RESISTANT CALLI
1. Transfer the roots onto Callus Inducing Medium (CIM) containing 100 ug/ml of Timentin and either 50 ug/ml of kanamycin or 10 ug/ml of phosphinothricin.
2. Seal the plates with double layers of parafilm and place them in a growth chamber for 4-5 weeks. Around 2 weeks after infection, you should be able to see small yellow calli appear.
TISSUE CULTURE MEDIUM
1. MS Basal Medium (for 1 liter)
4.32 g MS minimal salts (Gibco)
0.5 g MES
1 ml Vitamin stock solution (1000 X)
10 ml Myo-inositol stock solution (100 X)
10 g Sucrose
Adjust pH to 5.7 with 1 N KOH
7.5 g Bacto AgarAutoclave for 20-30 min
2. CIM (for 1 liter)
4.32 g MS minimal salts (Gibco)
0.5 MES
1 ml Vitamin stock solution (1000 X)
10 ml Myo-inositol stock solution (100 X)
20 g Glucose
1 ml IAA stock solution (1000 X)
0.5 ml 2,4-D stock solution (2000 X)
0.5 ml Kinetin stock solution (2000 X)
Adjust pH to 5.7 with 1 N KOH
7.5 g Bacto Agar
Autoclave for no more than 20 min
3. B5 Medium
Gamborg�s B5 medium (Gibco) (basal medium with minimum organics)
Dissolve the entire content from one bottle to make 1 liter medium
Adjust pH to 5.7 with 1 N KOH
7.5 g Bacto Agar
4. Stock Solutions :
Myo-inositol (100 X) 10 mg/ml
Vitamin (1000 X) 0.5 mg/ml Nicotinic Acid
0.5 mg/ml Pyridoxine
0.5 mg/ml Thiamine-HCl
IAA (1000 X) 5 mg/ml in H2O with a little help from KOH
2,4-D (2000 X) 1 mg/ml in H2O with a little help from KOH
Kinetin (2000 X) 0.6 mg/ml H2O with a little help from KOH
This work is made possible by a grant from the National Science Foundation's Plant Genome Research Program.
Questions or Comments regarding this web site should be addressed to Dr. Stanton Gelvin