Day 0
Plate pouring:
Autoclave the B and D medium and pour sterile glass plates (18 cm of diameter). Let them dry under bacterial hood and store them in the growth chamber (25°C, 80% humidity, dark).

Day 1
Start Bradyrhizobium japonicum culture:

In 200 ml of HM medium + chloramphenicol (20 μg/ml final concentration), 2 ml of a bacteria pre-culture are added.

The flask is shaking during 3 days at 30°C, 180 rpm.

Glycine max (Williams 82) seeds sterilization and sowing:
20% bleach 10’ (2 times)
Wash 3 times with sterile water
0.01M HCl (1 time)
Wash 3 times with sterile water
Let the seeds dry few minutes before sowing. 

The seeds are germinated on agar B and D medium. Each seed is separated from its
neighbors by around 2 centimeters. The plates are stored for 3 days in the growth
chamber (25°C, 80% humidity, dark). After 3 days germination occurs. Roots are around
1 to 2 cm long. 

Day 4
Bacterial inoculation:
After 3 days, the bacteria are washed in sterile water three times by centrifugation (4000 rpm, 10 min, room temperature) with gentle resuspension. After the last washing, the O.D.600nm is measured and sterile water is added to obtain an O.D.600nm of 0.8. The bacteria are sprayed on the 3 days old seedlings. As control, other plates are sprayed with water. After spray, the plates go back in the growth chamber for incubation (25°C, 80% humidity, dark) before to be harvested. We use a perfume sprayer for inoculation but any type of sprayer should work as long as it uniformly wets the plants.

Root Hair isolation:
The roots are cut from the seedlings and dropped directly into a liquid nitrogen container. The liquid nitrogen is stirred using a glass rod for 20 minutes. The stirring allows the separation of the roots hair cells from the roots. The stirring must not be too strong to avoid breaking the root tips. To isolate the root hairs, the liquid nitrogen is poured in a beaker through a sieve to retain roots. We use a metal strainer. The type you can buy at any store that sells kitchen supplies. To harvest the maximum amount of root hair, the liquid nitrogen container is rinse with liquid nitrogen 5 to7 times. Usually, from 1000 seedlings, 0.5 to 1 gram of root hair cells are harvested.


B and D medium:
Solution A (CaCl2 2 M) (per 100 ml)
22.2 g CaCl2
Solution B1 (KH2PO4 1 M) (per 100 ml)
13.6 g KH2PO4
Solution B2 (K2HPO4 1 M) (per 100 ml)
17.4 g K2HPO4
Solution C (Fe-citrate 0.02 M) (per 100 ml)
0.495 g Fe-citrate
Solution D (per 100 ml)
12.33 g MgSO4, 7 H2O (0.5 M)
8.7 g K2SO4 (0.5 M)
0.0338 g MnSO4.H2O (0.002 M)
0.0247 g H3BO3 (0.004 M)
0.0288 g ZnSO4.7H2O (0.001 M)
0.01 g CuSO4.5H2O (0.004 M)
0.0056 g CoSO4.7H2O (0.0002 M)
0.0048 g Na2MoO4.2H2O (0.0002 M)

Autoclave the 5 solutions.
In a 6L flask, add 2.25 ml of each solution (A, B1, B2, C and D) to 3 liters of water.
Complete the volume to 4.5 liters with water. Add 60 g of agar for 4.5 liters of medium. 

HM medium (per liter):
0.125 g Na2HPO4
0.25 g Na2SO4
0.32 g NH4Cl
0.18 g MgSO4.7H2O
0.004 g FeCl3
0.013 g CaCl2.2H2O
1.3 g HEPES
1.1 g MES
0.25 g Yeast extract
1 g D-Arabinose or L-Arabinose






Proteomics using 2-D SDS Polyacrylamide Gel Electrophoresis


2D gel electrophoresis: full details at


Trypsin digestion: Trypsin digestion is done before the mass spectrometer analysis, the gel plugs are washed 4 times 15 min. in wash solution (50% acetonitrile, 50 %, 50 mM ammonium bicarbonate). Gel plugs are then briefly dried in 100 % acetonitrile, incubated for 20 min. in 100 % acetonitrile, and completely air dried. They are rehydrated with a trypsin solution (Promega, 20 U) and incubated for 2 hours at 4°C. The trypsin solution is removed and replaced by 15 μl of 40 mM ammonium bicarbonate and 10 % acetonitrile. Trypsin digestion is performed overnight at 37°C. The samples are centrifuged for 30 sec. at 12000 g. The supernatant containing the peptides is transferred to a new tube containing 4 μl of extraction solution (60 % acetonitrile, 1% trifluoroacetic acid). Gel plugs are washed two times with extraction solution for 10 min. and the supernatants, after a centrifugation of 30 sec. at 12000 g, are collected and transferred to the tubes described above. Samples are then iophylized, reconstituted in 5 μl of 990/10 water/88% formic acid (V/V), ziptiped on a C18 column for purification and reconstituted in 10 μl of elution buffer (700/290/10 (V/V/V) acetonitrile/water/formic acid 88% elution buffer), frozen in liquid nitrogen.


Analysis of tryptic peptides: 0.5 μl digested purified peptides were mixed with the same volume of alpha-cyano-4-hydroxycinnamic acid (Fluka MS grade, Sigma-Aldrich, Saint Louis, USA) solution (5mg/ml in 500/380/20/100 acetonitrile/water/ 10% trifluoroacetic acid/100 mM ammonium dihydrogen phosphate). The sample/matrix (0.3 μl) mix was deposited on a stainless steel plate (ABI01-192-6-AB). The tryptic peptides were analyzed on an Applied Biosystems Inc. 4700 MALDI TOF/TOF MS in positive ion delayed extraction reflector mode with a 355 nm (200 Hz) laser. The instrument was calibrated with ABI peptide standards (4700 Mass standards kit, 4333604). Spectra were analyzed using the GPS Explorer software (v. 3.0) (Applied Biosystems) and the Matrix Science’s Mascot search engine ( against the NCBI Viridiplantae protein database. Search parameters included, a maximum of 150 ions per MS spectrum with a signal to noise ratio >20, a mass error of 0.1 Da for the monoisotopic precursor ions, a maximum of one allowed misscleavage by trypsin, an exclusion of peptide masses corresponding to the autolysis of the trypsin, carbamidomethylation of cysteines and methionine oxidation respectively as fixed and variable modifications.

Bradyrhizobium japonicum treatment of soybean composite plants

Manjula Govindarajulu and
Christopher Taylor

Non-tissue Culture Composite Plants

We have developed and tested the composite plant protocol for numerous species of plants including tomato, soybean, Medicago truncatula, green bean, broccoli, potato, sweet potato, sugar beet, tobacco, okra, and gourd.  The ease of handling, inexpensive materials needed, and speed for producing transgenic roots on wild-type shoots makes this composite plant system valuable to those who are in need of transgenic material for screening purposes. This protocol has been adapted from Collier et al., (2005) and Taylor et al., (2006).

  1. FibrGro® (also called rockwool): Catalog #14-2720 (Hummert International, Earth City, MO, USA).  Each cube should be cut into 1-1.5 cm cubes.  Use a pipette tip to make a hole into each FibroGro® cube.
  2. Plastic trays/containers: black flat trays with no holes: Catalog #11-3050 (Hummert International, Earth City, MO, USA). 
  3. Clear lids or propagation domes: Catalog #14-2568 (Hummert International, Earth City, MO, USA).
  4. Vacuum desiccator.
  5. Sterile Petri dishes (10X100mm). 
  6. Sterile 250 ml flasks.
  7. Sterile 15 ml culture tubes.
  8. 1801 deep pots: Catalog #116305-1 (Hummert International, Earth City, MO, USA).
  9. Soil:
    1. Metro Mix 360: Catalog #10-0356 (Hummert International, Earth City, MO, USA).
    2. Vermiculite (fine texture): Catalog #10-2200 (Hummert International, Earth City, MO, USA).
    3. Perlite: Catalog #10-1123 (Hummert International, Earth City, MO, USA).
  1. Prepare the following media:

Luria Broth (LB) medium (see end of protocol)
Nitrogen free plant nutrient solution (N- PNS; see end of protocol)
HM media (see end of protocol)



A.  Sterilization of soybean seeds using chlorine gas:
Because of potential contamination by endophytic or pathogenic fungi and bacteria in field grown soybean seed it is necessary to sterilize the seeds with chlorine gas prior to planting.
1.   Safety Note: be sure to perform this procedure in a chemical fume hood and not in a laminar flow hood.  
2.   Place a vacuum desiccator in a fume hood and place seeds in an open Petri plate. 
3.   In the fume hood add 200 ml of bleach to a 250 ml beaker.  Add 2 ml of concentrated HCl to the bleach. 
4.   Quickly place the beaker and the seed in the vacuum desiccator and close the lid. 
5.   Pull vacuum on the desiccator and shut off vacuum and close valve on desiccator.  Let seeds sit overnight.
6.   Carefully open up desiccator in the fume hood and quickly cover the seeds with the lid of the Petri plate.  The seeds are now ready for planting.

B.  Preparation of plant materials:
Sterilized soybean seed is planted in greenhouse prior to use.  Planting seeds in growth chamber and subsequent composite plant production was problematic due to rotting.  Greenhouse grown seed appears to be more vigorous and resist rotting.
1.   Plant seeds in steam-cleaned or autoclaved soil or sand.  Any size pot or flat will work provided enough moisture is retained for proper seed germination.
2.   Place pots or flats in the greenhouse and let seeds germinate and plants grow to desired size.  For making soybean composite plants we use plants that have 2-4 fully expanded trifoliate leaves. 
3.   When plants are two weeks old they are ready for use in making composite plants, although any age plant can be used. 

C.  Preparation of A. rhizogenes strain K599:
A. rhizogenes strain K599 is the best strain of A. rhizogenes for hairy root production in soybean.  Other strains have been tried but none have yield acceptable results.
1.   Streak A. rhizogenes strain K599 containing a binary vector from a glycerol stock onto LB medium containing the appropriate antibiotic for vector used.  Incubate at 28-30oC for 2 days.
2.   Transfer one Agrobacterium colony from plate into a sterile test tube containing 5 ml of LB broth plus antibiotic selection appropriate for vector used.
3.   Place test tube into a 30°C shaker/incubator for one day.
4.   The quantity of bacterial culture is determined by the number of shoots to be transformed. Each plant will require about 4 ml of final bacterial culture resuspended in autoclaved water.  Typically the amount of LB broth used is 1/3 that of autoclaved water.  This should make the final O.D. 600nm between 0.2-0.5.  (Example: we decide to transform 25 shoots, thus needing 100 ml of bacterial culture in autoclaved water.  We will then need to grow the cells in 100/3=33 ml of LB broth).
5.   Transfer 50 µl of full grown Agrobacterium culture from test tube into a sterile 250 ml flask containing the estimated amount of needed LB broth with antibiotic.
6.   Place flask onto a 30°C shaker/incubator overnight.
7.   Spin down the cells at 3,300g for 10 minutes.  Pour off the supernatant.  The pellet should be pinkish in color.
8.   Resuspend the cells with gentle agitation in the appropriate amount of autoclaved water to an O.D. 600nm =0.3.


D.  Inoculation and Co-culture:
It was found (accidentally) that drying is essential for getting good transformations and subsequent hairy root production. 
1.   Cut FibrGro® (rockwool) sheets to 1-1.5 cm cubes and autoclave.
2.   Put 3 to 5 cut rockwool cubes into an open Petri dish.  Use a 1 ml pipet tip to make a divot (or hole) in the cube.
3.   Add 4 ml of resuspended bacteria culture to each cube.  Place the open Petri dishes into a plastic tray.
4.   Cut the shoots (containing the apical meristem) off the desired soybean plants.  Make a slanting cut such that the area of infection is large. Cut the shoot in the middle of the internode region that is not quite fully expanded.
5.   Put one shoot into each hole of each cube.  Cover the tray with a clear lid and put it in a growth chamber or leave it on a well-lit bench top overnight for acclimation.  If placed in a growth chamber set conditions to: Temperature: 220C, Light: 15 micromoles/meter square/sec, and Humidity: 30%.
6.   On the second day remove the lid and let cubes dry out.  This will cause the tissue to become severely wilted (generally one to four days).  Check each plant daily.

E.  Explant recovery, acclimatization and care of transgenic plants:
Keeping light conditions low will reduce enhance hairy root production and reduce adventitious root formation.  Between 40-60% of the roots should be transgenic (if scored using a scorable marker such as green fluorescent protein).
1.   When plants are fully wilted, water cubes with deionized water until cubes become saturated. 
2.   Place lids back on trays.  Within several hours the plant shoots should recover from wilting.       
3.  After a week, place the composite plants in to trays containing vermiculite:perlite (3 parts:1 part) wetted with N- PNS. Cover the pots with clear plastic domes and place them in a growth chamber with the following conditions: Temperature: 220C, Light: 15 micromoles/meter square/sec, and Humidity: 30%). Water every other day, alternating water with N- PNS.
4. After one week, transfer the pots to a growth chamber with the following conditions: Temperature: 260C, Light: 200 micromoles/meter square/sec, and Humidity: 60%.

F.  Bradyrhizobium japonicum treatment: 
After a total of two weeks in vermiculite: perlite, the plants have enough roots for B. japonicum inoculation.  Keep the plants warm (between 25oC and 28oC) to ensure proper nodulation.

  • Grow B. japonicum in starter culture (3 mL or 10 mL) for three days. Inoculate on to larger culture – depends on the number of plants you have – and let grow for another three days (usually to OD600nm = 0.5 or 1).
  • Spin B. japonicum cells down at 7,000g, 10oC for 15 minutes.  Resuspend pellet with sterile water and spin down the cells.  Finally, resuspend in N- PNS to OD600nm = 0.08.
  • Add 10 ml of this B. japonicum suspension to each pot.
  • In about two weeks, you will start to see nodules form.
  • Start counting nodules 4 weeks after the B. japonicum treatment.
  • Carefully pull plants out from the vermiculite:perlite mix and wash roots and put in a beaker of water.
  • Cut each root and put in a 15 ml tube in water.
  • Run all tubes under the UV microscope and score for transgenic root production (usually using GFP as a scorable marker).
  • Count nodules on each root and document.


Media for use in composite plant protocol



Luria Broth (LB) medium:
10 g Bacto Tryptone
5 g Bacto Yeast Extract
10 g NaCl
Suspend in 1 liter of MilliQ water.
Autoclave  for 30 minutes.
Media can be stored at room temperature.


HM Media:



0.125 g




0.25 g




0.32 g

MgSO4 * 7 H2O


0.18 g

Yeast extract


0.25 g



1 g



1 g

FeCl3 (1 mM)


0.004 g

CaCl2 * 2 H2O


0.013 g




1.3 g




1.1 g




Adjust pH 6.6 with NaOH.
Take to 1 liter and autoclave for 30 minutes.
Media can be stored at room temperature.
Add sterile Chloramphenicol (20mg/mL = final concentration) prior to use.


Nitrogen Free Plant Nutrient Solution for Soybean (N-PNS solution):

1 Liter (Stock)              Working solution (per Liter)

MgSO4 . 7H2O                     61.5 g                                      2 mL
CaCl2 . 2H2O                       73.5 g                                      4 mL
K2HPO4 . 3H2O                   34 g                                         1 mL
K2SO4                                    55 g                                         4 mL
FeCl3 . 6H2O/                       2.45 g                                      2.5 mL
Micronutrients                     see below                                 1 mL

Micronutrients                                                           500mL (Stock)

H3BO3                                                                        0.071 g
MnSO4 . H2O                                                             0.0385 g
ZnSO4 . 7H2O                                                           0.08625 g
CuSO4 . 5H2O                                                          0.0185 g
NaMoO4 . 2H2O                                                        0.012 g
CoCl2 . 6H2O                                                            0.00125 g
NiSO4                                                                         0.0005 g



Taylor, C.G., Fuchs, B., Collier, R., and Lutke, K., (2006) Generation of composite plants using Agrobacterium rhizogenes.  In Agrobacterium Protocols ed. Wang, K. Methods in Molecular Biology 343, Humana Press, pp 155-167.

Collier, R., Burgwyn, B., Walter, N., Lutke, K., and Taylor, C.G. (2005) Ex vitro composite plants: an inexpensive, rapid method for root biology Plant Journal 43:449-457.

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