Strong Allelopathic Activities of Leaves and Cultured Cells of &i&Spiraea thunbergii&/i& Assayed by the Protoplast Co-Culture Method with Digital Image Analysis: Evaluation of &i&Cis&/i&- and &i&Trans&/i&-Cinnamic Acid as Allelochemicals

摘要

The inhibitory allelopathic activities of leaves and leaf-origin suspension cultured cells of Spiraea thunbergii , and putative allelochemicals, cis - and trans -cinnamic acid, were investigated by the protoplast co-culture method with digital image analysis (DIA-PP method) using lettuce as a recipient. The optimal conditions of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) for the cell division of S . thunbergii protoplasts were first examined using 50 μL liquid MS basal medium containing 3% sucrose, and 0.8 M mannitol in a 96-well culture plate. The hormonal condition for co-culture, 1 μM 2,4-D plus 0.1 μM BA, which was optimal for lettuce protoplast growth, was sub-optimal for S . thunbergii protoplasts. Effects of co-culture on the three stages of lettuce protoplast growth, i.e. , cell wall formation, cell division, and yellow pigment accumulation, were examined. Protoplasts of leaf and suspension cells of S . thunbergii strongly inhibited lettuce protoplast growth at the cell division stage (100% inhibition at 80 - 100 × 10~(3) mL~(-1)), but not so much at the other two stages. Both cis - and trans -cinnamic acid, showed the strongest inhibition at the cell wall formation stage, and 100% inhibition at the cell division stage at 100 μM. These results were compared with those obtained in a lettuce seedling growth test, using different allelopathic plants, and their allelochemicals were studied by the DIA-PP method.