Symbiotic Fungi: Principles and Practice (Soil Biology)

376 A. Baldi et al.
3. Place one explant in one Petri dish containing 15 ml of MS medium (Murashige
and Skoog 1962) solidified with 1% agar and supplemented with 0.4 mg l 1
NAA (naphthalene acetic acid) for callus initiation using a sterile forceps.
4. Cover the petri dish with lid and seal it with parafilm.
5. Transfer Petri dishes in a culture room at 25 2 C under 16 h/8 h light/dark
cycle with a light intensity of 1,200 lux.
6. Observe the callus initiation.
23.2.3 Initiation of Suspension Cultures
23.2.3.1 Inoculum Preparation
1. Transfer fresh and friable cells (20 days old) from callus culture (5 g l 1 on dry
cell weight basis) into a 250 ml Erlenmeyer flask containing 50 ml MS media
containing 0.4 mg l 1 NAA with the help of sterile spatula over flame.
2. Plug the culture tubes with sterile cotton plug over flame.
3. Place inoculated flasks on a gyratory shaker rotating at 125 rpm in a culture room
at 25 2 C under 16 h/8 h light/dark cycle with a light intensity of 1,200 lux.
4. Allow the cells to grow in suspension culture for 12 days.
5. Centrifuge the grown cells in sterile centrifuge tubes at 5,000 rpm for 10 min.
6. Decant the spent media and use cells to inoculate suspension cultures for
elicitation setup.
23.2.3.2 Development of Suspension Culture
1. Transfer these cells (5 g l 1 on dry cell weight basis) into a 250 ml Erlenmeyer
flask containing 50 ml MS media containing 0.4 mg l 1 NAA with the help of
sterile spatula over flame.
2. Place inoculated flasks on a gyratory shaker rotating at 125 rpm in a culture room
at 25 2 C under 16 h/8 h light/dark cycle with a light intensity of 1,200 lux and
allow the cells to grow for 12 days.
23.3 Development of Elicitors
23.3.1 Preparation of Elicitors
1. Transfer cells of arbuscular myccorhiza like fungus, Piriformospora indica, to
a 250 ml Erlenemeyer flask containing 50 ml of MYPG media of following
composition: