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

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 × 103 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.