Methodological setup to study allelochemical translocation in radish seedlings

[Ring-U14C] p-hydroxybenzoic acid (POH) translocation during radish germination and early seedling growth was studied to compare two culture conditions (sterile/non-sterile) and two modes of POH extraction (ethanol/oxidizing). Quantification of POH in organs was performed by grinding them in a mortar with ethanol or by combusting them in a biological oxidizer. Comparison of these extraction methods revealed that the oxidizer provides higher POH concentrations than grinding. Uptake and translocation of POH into radish seedlings occurred in the first 24 hr with the highest accumulation in cotyledons. POH in cotyledons ranged from 8 to 12.4 mumol g-1 FW. Two forms of POH were found in seedlings, an ethanol-soluble one and a "bound" one. After 96 hr incubation, half of the recovered POH was "bound" in roots and hypocotyls. POH recovery in seedlings was also higher under axenic rather than non-axenic conditions. POH was degraded into 14CO2 by microorganisms under non-sterile cultures, its concentration in culture medium decreased from 10(-3) to 10(-4) M between the first and the fourth day of incubation. Choice between sterile and non-sterile culture conditions as a method depends on particular research objectives. Non-sterile methods can be used to reflect natural processes whereas sterile methods can be used if the objective is to determine allelochemical penetration and biological effects on target plants.