The activity of the xantho-oligosaccharides inducing phytoalexin accumulation in soybean cotyledon tissues was determined using the soybean cotyledon bioassay as
described by Hahn et al. [24] with some modifications.
After soaked in sterile distilled water for 24 h, the seeds of the soybean were placed in trays containing wet filter paper and covered with the moist gauze.
The seedlings were watered daily with tap water.
A photoperiod regime of 8 h of light and 16 h of darkness was used.
The temperatures during the light and dark cycles were 25 and 20 8C, respectively.
Cotyledons were detached from 9- to 11-day-old seedlings at the time when the primary leaves had just expanded.
Detached cotyledons were surface sterilized with 0.5% sodium hypochlorite for 5 min and then extensively rinsed with sterile distilled water.
Tissue sections with 1 mm deep and 6 mm length were removed from the surface of the cotyledons. Wounded cotyledons were transferred to sterile Petri dishes (10 cotyledons per dish), and 10 ml of sample solution was applied to the entire wound surface of each cotyledon.
Three dishes, each containing 10 cotyledons, were used for each sample analyzed.
The cotyledon dishes were incubated in the dark at 25 8C for 24 h.
After incubation, all 10 cotyledons were transferred to a beaker containing 10 ml of distilled water and shaking for 1 min.
The optical density of these solutions was read at 286 nm.