Orobanche germination assay on filter paper

Surface sterilized seeds are vacuum dried on a Bichner apparatus and then sprinkled on four 5 cm diameter glass-microfiber filter discs (Whatman GF/A paper) wetted with 1.0 ml sterilized water and placed in 5 cm diameter Petri dish.

The Petri dishes are sealed with Parafilm, packaged in aluminum foil and put in a growth chamber (23C, 14h light of 100 mE m2s-1) for a preconditioning period.

Two weeks later, Petri dishes are opened and sixty surface sterilized Arabidopsis seeds are placed on each 5 cm glass-microfiber disc. A positive control is prepared by not placing Arabidopsis seeds on the GF/A discs but applying 0.6 ml aqueous solution of the synthetic strigol analogue GR24 to each Orobanche-inoculated GF/A disc (5 ppm for O. aegyptiaca, O. ramosa and O. minor, 10 ppm for O. crenata and O. cumana). A negative control consists of treating the Orobanche seeds with sterilized water instead of the GR24 solution.

All Petri dishes are resealed with Parafilm and returned to the growth chamber. Fourteen days after treatments, Orobanche seed germination rate is determined under a stereoscopic microscope by counting the number of seeds with and without an emerged radical.