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

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.