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Abstract
Sampling procedures of the donor material, conditions of the assay, and selection of test organism influence the sensitivity of a bioassay. Autotoxicity affects success of alfalfa (Medicago sative L.) reseeded after old alfalfa. Root length is a more sensitive parameter to autotoxin than germination or hypocotyl length. Our specific objective was to improve the sensitivity of an alfalfa seedling bioassay for a better understanding of the autotoxicity mechanism. Using a petri-dish assay with imbibed seed, we found extract of alfalfa leaves was more autotoxic than either that of stems or seed. Extracts from oven-dried samples were stronger than those from freeze-dried samples. Sterilization through membrane filtering or autoclaving did not alter autotoxicity bioassay. Root length at 25 or 50 seed/plate was more sensitive to the extracts than that at 100 or 200 seed/plate. Roots from older seedlings were more tolerant to the extracts than did those from seed or younger seedling and had more branch roots to escape the autotoxin(s). These results suggest that extracts from leaf samples that oven-dried and sterilized by filtering or autoclaving can improve the consistency and precision of bioassay. The bioassay was suitable with 25 to 50 seed per petri dish to minimize interaction on interference (e.g., competition and autotoxicity from testing seed itself). The bioassay can be improved by using very young roots from imbibed seed which are more sensitive to the autotoxin.