Modified from Kevin Lutke, Christopher G. Taylor and Manjula Govindarajulu
Materials and Methods
– Media and Antibiotics
10 g Bacto Tryptone
5 g Bacto Yeast Extract
10 g NaCl
Suspend in 1 liter of MilliQ water.
Autoclave (30 min.)
Store at room temperature
¼ MS – liquid and solid
Dissolve 1.1g Murashige and Skoog Basal Medium powder (Phytotechnology Laboratories M524) into MilliQ water
Adjust pH to 5.8 using 1 M KOH
Take to 1 liter
Autoclave (20 min.)
Store at 4°C
For solid media add 750mg/L MgCl2 before adjusting pH and 3g/L Phytagel (or Gelrite) before autoclaving.
Carbenicillin: used for curing plant tissue of agro
Dissolve 500mg/mL to make to make a 1000x stock solution
Store at –20°C
A. Soybean seeds sterilization using chlorine gas
- ~100 seeds are placed in a 90 x 15mm plate about 5 plates of seed can be done at one time.
- Place plates of seeds in a vacuum chamber, in a fume hood.
- Using a 250-350mL glass beaker, fill with 200mL of 100% Clorox bleach.
- Using a 5mL pipette, carefully add 5mLof concentrated hydrochloric acid (HCl) slowly, a few drops at a time (you will see the bubbling when the HCl is added).
- After the addition of the HCl, stir using the pipette to mix and discard the pipette.
- Close the chamber and let sit for a day (18-24 hours).
- After sterilization aseptically seal plates with seeds with parafilm and store
B. Soybean Seed Germination (7 days)
- After the seeds are sterilized they are then ready to be placed on media.
- Prepare plates (~5-8 seeds/plate) of ¼ MS solid media.
- Using aseptic technique, transfer individual seeds and press slightly in the media to keep them from moving around.
- After the seeds are all placed, wrap the plates with parafilm and place in 18hr:6hr light:dark conditions in a growth room at 25°C.
- Germinate seedling for 7 days.
C. Agrobacterium rhizogenes initiation (1-2 day process)
- Culture of A. rhizogenes can be grown for 24 hours or 48 hours depending on the desired amount needed.
- Twodays before inoculations, agro is initiated in a 10mL culture of LB plus antibiotics. This is done by spiking the LB with cultures from stored glycerol stocks.
- The spiked agro culture is placed on a 28°C shaker. After one day growth, the cultures should be used to respike a fresh 10mL of LB plus antibiotics and grown overnight on a 28°C shaker.
- On the day of transformation, spin the agro culture down at 4,000g for 10 minutes to pellet the bacteria. The pellet should be slightly pink, and not brown (E. coli or other contaminates usually produce brown pellets).
- Resuspend the pellet is in ¼ MS liquid media. A typical dilution is 1:10 for a two-day culture, or a 1:5 for a one-day culture. This should yield an OD600nm (optical density) of ~ 0.2-0.3. It may be a good idea, after the two day growth, to resuspend in 50mL of ¼ MS and take an OD600nmwhich should be about 0.5 to 0.6. After that, the cultures can then be diluted accordingly (usually another 1:2) to achieve a final OD600nm 0.2-0.3.
D. Explant Excision and Inoculation
- After 7 days of germination and the agro is prepared, the cotyledons are ready to be cut. The cut is made between the hypocotyl and the ½ way point of the cotyledon. The seed coat is removed if it hasn’t fallen off yet. The cut will remove the hypocotyl and about 25% of the cotyledon, leaving 75% of the cotyledon for transformation.
- In a 90 x 15mm petri plate place the cotyledons into about 30mL of agro solution. This can be done to as many cotyledons as needed.
- When the cotyledons are in the agro solution place the plates in a vacuum chamber for “vacuum infiltration”.
- Pull a vacuum for about a minute and then close the valve. Let sit for 20 minutes.
- After 20 minutes release the vacuum, (preferably in a laminar flow hood).
- Remove the agro solution using a sterile 25mL pipette. Place the cotyledons flat side upon a sterile 70mm sterile filter paper in a 90 x 15mm petri plate. If necessary add 1-2 ml of ¼ MS liquid media to the filter to help prevent desiccation, if the filter paper is too wet and the liquid pools at the bottom of the plate when tilted, remove the excess liquid with a sterile pipette tip.
E. Explant Co-culture and Wash Step
- Wrap the plates in parafilm to prevent desiccation and place in a lighted growth chamber and incubate for 3 days (18hr:6hr light:dark, 23-26°C).
- Remove the cotyledons from the filter paper and place in a 90 x 15mm plate. Add 20-30mL of ¼ MS liquid media plus Carbenicillin (500mg/L) and wash cotyledons for 30 minutes.
- Remove cotyledons from the liquid, and force each cotyledon into ¼ MS solid media plus Carbenicillin (500mg/L), with the cut surface facing up out of the media. Place 6 cotyledons per plate. Wrap plates in parafilm.
- After about 7 days post-inoculation small calli will start to appear on the cut surface. After 14 days post-inoculation roots will appear from calli, most will be hairy roots though some adventitious roots may arise. Hairy roots can be identified by the presence of a scorable marker (dsRed) usually incorporated into the binary plasmids’ T-DNA.
F. Hairy Root Production
- After about 18-25 days post-inoculation, hairy roots can be isolated and removed from the cotyledons. At this point in time, dsRed positive roots can be isolated and cultured individually. Cut individual root (each is an independent event) from the cotyledon and place on modified Whites solid media (see protocol below) plus carbenicillin 500mg/L. Wrap the plates in porous tape (Micropore) and place in humid boxes in the dark at 26°C.
- After about 2-3 weeks, the roots can be subcultured by trimming off about 3-4cm long lateral roots, or from the primary root tip and placing on the modified Whites solid media. Carbenicillin should be used for the first 3-4 subcultures to insure that the agro does not grow, after which the carbenicillin can be removed from the media.
- Most roots survive and the healthier roots will be white and grow rapidly. Roots can be maintained for several months if they are healthy and clean.
Each individual dsRed positive root emerging from the cotyledon is an independent transformation event and should be labeled as such (Root 1, Root 2, Root 3, etc). When culturing lateral roots from the “parent root,” label them Root 1a, Root 1b, Root 1c, etc.