Application of the protoplast co-culture method for evaluation of allelopathic activities of volatile compounds, safranal and tulipalin A

Abstract The effects of two plant volatile compounds (VOCs), safranal and tulipalin A were investigated using the protoplast method with digital image analysis, which was developed to investigate allelopathic activities of plants at the cellular level in a 50 μL liquid medium. Both VOCs showed inhibition on lettuce protoplasts growth at all three stages, i.e., the cell wall formation stage, the cell division stage, and the yellow colour accumulation stage. Among the three stages of protoplast growth, differences of inhibition patterns were observed. Inhibitions at the cell wall formation stage were stronger than those at the latter two stages in both VOCs. Tulipalin A showed higher inhibitory activity (total inhibition at 100 μM) than that of safranal (44% inhibition at 1 mM) at cell division stage. They were also compared with the direct exposure method in an enclosed vial using germinated lettuce seedlings, which was developed to investigate allelopathic activities of VOCs at plant level. Tulipalin A showed less inhibitory activity on radicle growth of lettuce, than that of safranal reported. Inhibition on hypocotyl growth was stronger than that on radicle in both VOCs. The causes of the differences between two VOCs and two bioassay methods, and the application of the protoplast method, were discussed for clarifying the contribution of VOCs as allelochemicals at cellular level.