Symbiotic Fungi: Principles and Practice (Soil Biology)

24 Auxin Production by Symbiotic Fungi 385
In addition, roots may be harvested from phytagel without damage by dissolving
the solid medium in three volumes of 100 mM sodium citrate buffer pH 6.
Remark: If buffering the pH of the medium is required, remember that an acidic
medium will facilitate IAA uptake in the seedlings better than a neutral one,
because of passive diffusion of of the protonated IAAH through the cell walls
(Yang et al. 2006).
Remark: In mycorrhizal research, lower sucrose concentrations are often
recommended to improve the colonization of the root by the fungus. We have
tested sucrose concentrations in a range between 0.5 and 2%, and obtained similar
results with regard to root branching of A. thaliana inoculated with Piriformospora
indica.
Remark: Round Petri dishes (Ø 9.0 cm) can be used instead of square ones, but
the development of the roots can then only be clearly observed in the middle of the
plates, especially in the controls where the roots tend to be longer.
24.3.2 Inoculation of the Fungus
Place mycelial plugs from the growing margin of a colony below the seeds, as
indicated in Fig. 24.1.
24.3.3 Adding Arabidopsis Seeds to the Petri Dish
Sterilize seeds as follows:
l 70% ethanol, 1 min
l 20 min in 1% sodium hypochloride
l Rinse three times with sterile water
Transfer sterilized seeds to a sterile wetted filter paper in a round Petri dish and if
needed stratify them (2 days at 4 C) to synchronize germination (may be necessary
especially for some mutants of A. thaliana).
To transfer seeds onto the agar, take them one by one with the tip of a sterile
toothpick and gently place them at the surface of the agar, as illustrated in Fig. 24.1.
Alternatively, suspend sterilized seeds in 0.1% sterile water agar and pipette seeds
one by one onto the rim of the agar slope.
Remark: We recommend a maximum of 10 seeds of A. thaliana per Petri dish if
precise microscopic observations need to be made on root branching and root
length. The bioassay can be followed up and the root branching quantified until
the rooting system becomes too entangled to distinguish primary from secondary
roots. This time period depends on the fungus; for truffles (e.g. Tuber melanosporum
or T. borchii) we were able to monitor root development of A. thaliana in
this system for 15 days.