Polyethylene bag assay of Orobanche parasitism of Arabidopsis

This is a non-destructive assay useful for monitoring multiple stages of parasitism.

Orobanche and Arabidopsis seeds are surface-sterilized by soaking them in 70% ethanol for 1 minute and then in 1% sodium hypochlorite+0.1% ‘Tween 20’ for 15 minutes. The seeds are washed 3 times with sterile water.

Surface-sterilized Orobanche seeds (0.01g) are sprinkled onto sterilized moist 14 by 12 cm glass-fiber sheets and placed in the 23°C growth chamber. 

Arabidopsis seeds are placed on ˝ strength MS agar in a 5 cm diameter Petri dish and put into the 23°C growth chamber. After two weeks, 6 seedlings are extracted from the agar and mounted on the top of each glass-fiber sheet. Each sheet is placed in a clear 25 by 18 cm polyethylene bag that is placed upright in a black box so that plant roots are in the dark box and their shoots project into the light above the box. Plants are covered with another polyethylene bag to retain moisture and the box is placed in the 23°C growth chamber. Bags are replenished with Hoagland nutrient solution as needed.

Observations regarding Arabidopsis growth and Orobanche seed germination, radicle attachment and development are conducted periodically on either 2 by 2 cm root sections or on the whole root system using a stereoscopic microscope (X10-X60).