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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