Materials and Reagents

Germination plate (1% agar in sterilized water)
B5 salt
Agar
Sucrose
Gamborg’s vitamin solution (Sigma-Aldrich, catalog number: G1019 )
Meropen (Dainippon Sumitomo Pharma)
LB medium
Sterilized water
Co-cultivation medium (see Recipes)
Hairy root elongation medium (see Recipes)

Equipment

Clean bench
L. japonicus growth facility
Surgical knife
Sterilized dish (9 cm in diameter)
Sterilized filter paper (7-8 cm in diameter)
Sterilized square dish (10 x 14 cm)

Procedure

A. Plant growth

Sandpaper the surface of L. japonicus Gifu or MG-20 seeds, and then incubate them in 2% sodium hypochlorite solution for 5 min. Wash the seeds several times with sterilized water and incubate the seeds overnight in the sterilized water.
Surface-sterilized seeds are germinated and grown in the germination plate.
Place the plate vertically in a growth cabinet (For Gifu, 23 °C 24 h dark for first 3 days and 23 °C 16 h light/8 h dark for next 2 days; For MG-20, 23 °C 24 h dark for first 2 days and 23 °C 16 h light/8 h dark for next 2 days).

B. Culture of Agrobacterium

Streak A. rhizogenes harboring the desired construct on LB plate with appropriate antibodies for 2 days at 28 °C, then spread bacteria all around sterilized dish (9 cm in diameter) and incubate for 1 day.

C. Infection of A. rhizogenes with L. japonicus

Collect the bacteria with bacteria spreader from LB plate and suspend 6 ml sterilized water.
Set a sterilized filter paper in a new dish, and saturate it with bacterial suspension by pipetting.
Place the juvenile plants on the saturated filter paper, and cut at the middle of the hypocotyl with surgical knife (Figure 1).
Transfer the seedlings of shoot side onto co-cultivation media (cut end is need to be about 1 mm in depth from agar surface), and place the plate horizontally in a growth cabinet (23 °C 24 h dark) for 1 day.
Place the plate vertically and incubate at 23 °C (16 h light/8 h dark) for 5 days.

D.Induction of hairy roots

Transfer the plants onto hairy root elongation media and incubate vertically in a growth cabinet (23 °C 16 h light/8 h dark).
After 10-14 days, the hairy roots should be approximately 2-5 cm in length. Pick up plants with transgenic roots expressing florescent proteins for further analysis (Figure 1).

Figure 1. Hairy root transformation in L. japonicus

Recipes

Co-cultivation medium
1/2x B5 salt
1/2x Gamborg’s vitamin solution
1% agar
Maintain pH with KOH at pH 5.5, pour into a square dish
Mix all components except for Gamborg’s vitamin solution, and autoclave the mixture, and then add Gamborg’s vitamin solution.
Hairy root elongation medium
1/2x B5 salt
1/2x Gamborg’s vitamin solution
12.5 μg/ml meropen
1% sucrose
1% agar
Maintain pH with KOH at pH 5.5, pour into a square dish
Mix all components except for Gamborg’s vitamin solution and meropen, and autoclave the mixture, and then add Gamborg’s vitamin solution and meropen.

Acknowledgments

This work was supported by MEXT/JSPS KAKENHI, Japan (22870035, 23012038, 25114519 to Takuya Suzaki).

References

Kumagai, H. and Kouchi, H. (2003). Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. Mol Plant Microbe Interact 16(8): 663-668.
Okamoto, S., Ohnishi, E., Sato, S., Takahashi, H., Nakazono, M., Tabata, S. and Kawaguchi, M. (2009). Nod factor/nitrate-induced CLE genes that drive HAR1-mediated systemic regulation of nodulation. Plant Cell Physiol 50(1): 67-77.
Suzaki, T., Yano, K., Ito, M., Umehara, Y., Suganuma, N. and Kawaguchi, M. (2012). Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response. Development 139(21): 3997-4006.