In vitro Bioassay of Allelopathic Activities of Soybean, and Three Isoflavones, Using Protoplast Co-culture Method with Digital Image Analysis

Abstract

Inhibitory allelopathic activities of dried leaves of potted plants and protoplasts of Glycine max (soybean) cv. Okuhara-wase were studied using two in vitro bioassay methods. Strong inhibition (90%) of growth of recipient lettuce root was obtained with 50 mg soybean leaves using the lettuce seedling growth test (sandwich method), and moderate inhibition (65%) of lettuce protoplast division was obtained with 100 × 103 mL-1 protoplasts of etiolated soybean seedlings using the protoplast co-culture method with digital image analysis (DIA-PP method). Three isoflavones, genistein, genistin (genistein-7-O-glucoside), and daidzin (daidzein-7-O-glucoside) were investigated as putative allelochemicals using the DIA-PP method. The three isoflavones at 100 µM showed early crystal accumulation in lettuce protoplasts and inhibited lettuce protoplast division by 100%. Genistein showed the strongest inhibition. The results were compared and discussed with the previous reports on the invader leguminous plant, Kudzu, and two isoflavones, daidzein and Kudzu-specific puerarin (daidzein-6-C-glucoside), and other allelopathic plants and their putative allelochemicals.

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

“…Strong inhibition was observed at the cell division stage (100% inhibition by 100 × 10 3 mL -1 ), slight stimulation was observed at the cell wall formation stage, and intermediate inhibition was observed at the yellow pigment stage. On the other hand, the inhibitory allelopathic activity of neoxanthin was very high at the yellow pigment accumulation stage (Sasamoto et al, 2021), which can be partly explained by the yellow pigment, a carotenoid, accumulating in lettuce protoplasts (Sasamoto et al, 2017a), which might be inhibited by the exogenously supplied carotenoid.…”

“…Digital image analysis with the protoplast co-culture method (DIA-PP method), was further developed to examine the effects of allelochemicals on the yellow pigment accumulation, which is specific to recipient lettuce protoplast growth at the late stage of co-culture (Sasamoto et al, 2017a, b). Many plant species and allelochemicals have been examined using the DIA-PP method (Sasamoto et al, 2022).…”