Strong Allelopathic Activity in Purple Leaves of Transgenic Spiraea cantoniensis Containing Cyanidin 3-Glucoside Assayed by the Protoplast Co-culture Method with Digital Image Analysis

The protoplast co-culture method with digital image analysis (DIA-PP method), was used for in vitro bioassay of allelopathic activity in the leaves of in vitro cultured plants of Spiraea cantoniensis. Purple leaves of transgenic S. cantoniensis, containing an anthocyanin, cyanidin 3-glucoside showed strong inhibitory allelopathic activity, 100% inhibition of recipient lettuce protoplast division by 80 × 103 mL-1 of co-cultured protoplasts, while green leaves of non-transgenic S. cantoniensis showed only 20% inhibition. Cyanidin 3-glucoside which inhibited lettuce protoplast division 80% at 100 µM was evaluated as an allelochemical of transgenic S. cantoniensis. Inhibition was stronger at the cell division stage of lettuce protoplast growth than that at the other stages, i.e., cell wall formation stage and yellow pigment accumulation stage. The results were discussed comparatively with the strong inhibitory activity of non-transgenic S. thunbergii and other allelopathic plant species containing different anthocyanin or carotenoid pigments as putative allelochemicals.