Abstract
This is a protocol to produce stable transgenic plants in Lotus japonicus, which is established based on methods previously reported (Handberg and Stougaard, 1992; Stiller et al., 1997; Thkjaer et al., 1998) with some modifications. In this protocol, hygromycin is used to select transgenic plants.
Materials and Reagents
Equipment
Procedure
A. Plant growth
B. Culture of Agrobacterium
Streak A. tumefaciens 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. We use AGL1 as agrobacteria strain.
C. Infection of A. rhizogenes with L. japonicus
D. Callus induction
E. Shoot induction
Transfer the calluses onto callus medium without hygromycin B (about 1 cm thick) and place the plate in a growth cabinet (23 °C 16 h light/8 h dark) for no longer than 7 weeks. Transfer the calluses onto new medium every 5 days (Figure 3). Shoots start to appear in about 3 weeks. Figure 3. Shoot induction
F. Shoot elongation
When shoots start to appear, transfer the calluses onto shoot elongation medium (about 1 cm thick) and place the plate in a growth cabinet (23 °C 16 h light/8 h dark) for no longer than 6 weeks. Transfer the calluses onto new medium every 5 days (Figure 4). Figure 4. Shoot elongation
G. Root induction
1. When shoots become about 1 cm in length, cut off the shoots from the calluses and stick them shallowly into root induction medium (about 1 cm thick). 2. Place the plate in a growth cabinet (23 °C 16 h light/8 h dark) for 10 days (Figure 5). Figure 5. Root induction
H. Root elongation
Transfer and stick the shoots into root elongation medium (about 3 cm thick) and place the magenta box in a growth cabinet (23 °C 16 h light/8 h dark) until their root length become about 2-3 cm (Figure 6). Figure 6. Root elongation
I. Naturalization
Transplant the plants into 100% vermiculite and grow them with high humidity at least first 1 week (23 °C 16 h light/8 h dark), and keep growing them for further analysis. To keep high humidity, we cover the pots of plants with Saran wrap.
Recipes
Acknowledgments
This work was supported by MEXT/JSPS KAKENHI, Japan (22870035, 23012038, 25114519 to Takuya Suzaki).
References
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Phytagel should be added the medium before autoclaving. The pH of the medium does not affect the solidification in this case.