Sterilize the surface of tomato seeds. Soak the tomato seeds in tap water
for one night. Immerse the seeds for 10 minutes in a 4-5% sodium
hypochlorite solution (HClO). Stir the solution continuously with a
magnetic stirrer. Pour off the sodium hypochlorite solution as soon as the
seed surface turns yellow. Rinse the sterile seeds in sterile tap water.
Place the sterile seeds on 2% water agar and allow them to germinate on sterile glass or plastic petri dishes.
Place the seedlings on water agar and cut the roots as soon as they reach a length of 1 cm. The cut roots must be transferred to the nutrient medium,
Gamborg B5 with 20 g/l of sucrose.
Transfer the root segments to the petri dishes containing the nutrient medium and seal with Parafilm and incubate at 25°C.
Surface sterilize fresh, full grown Meloidogyne eggs masses collected from the roots and incubate the egg masses in a 1% sodium hypochlorite
solution, in a vacuum dessicator.
Rinse the sterile eggs, and place them in sterile water, on the tomato roots and incubate the dishes at 230C and carry out regular observations.
Burrowing Nematodes
Select one or two thick fresh carrots, wash thoroughly and wipe with
tissue paper.
Dip the carrots in ethanol and flame them until the peel becomes dry and black under a laminar flow system.
Cut the tips and peel the carrot deeply with a sterile blade or peeler.
Cut them into discs of 1 cm thickness and place one or two discs in a sterile Petri plate.
Seal the Petri plates with parafilm, place them in plastic boxes and store at 280C in an incubator.
Collect R. similis from infested black pepper roots by hand picking.
Add one ml of 6000 ppm streptomycin sulphate to a sterile test tube containing nematodes in 2 ml sterile water and leave them overnight.
Collect R. similis from infested black pepper roots by hand picking.
Remove streptomycin sulphate using a sterile pipette and rinse with sterile water 2-3 times.
Transfer the above surface sterilized nematodes using a sterile pipette to the edge of the carrot disc.
Incubate the Petri plates at 280C in the dark for 20-30 days.
Under the authority of IISR, developed by
the Bioinformatics Centre
Protocols (Phytophthora
Related) 