TITLE:
Evaluation of Isoflavones as Allelochemicals with Strong Allelopathic Activities of Kudzu Using Protoplast Co-Culture Method with Digital Image Analysis
AUTHORS:
Kentaro Kobayashi, Hamako Sasamoto, Yutaka Sasamoto, Akifumi Sugiyama, Yoshiharu Fujii
KEYWORDS:
Allelopathy, Bioassay, Daidzein, Isoflavone, Protoplast Culture, Puerarin
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.12 No.3,
March
30,
2021
ABSTRACT: The inhibitory
allelopathic activity of Pueraria montana (Kudzu),
and activities of two putative allelochemical isoflavones, puerarin and daidzein,
were evaluated using the protoplast co-culture method with digital image
analysis using lettuce as a recipient (DIA-PP method). Cotyledon protoplasts of Kudzu were
isolated using Cellulase R10 and Driselase 20 in 0.6 M mannitol solution.
Optimal hormonal condition and density for growth of Kudzu protoplasts were
surveyed. Medium for co-culture of Kudzu or isoflavones with lettuce
protoplasts was 50 μl liquid MS basal medium containing 1 μM
2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose, and 0.4 M or
0.6 M mannitol. Protoplast division of lettuce was strongly inhibited by Kudzu
at a low density (104/ml). Slightly less inhibition by Kudzu on cell
wall formation and yellow pigment accumulation stages of lettuce growth was
also observed. Puerarin did not inhibit the growth of lettuce protoplasts at
three growth stages but slightly stimulated growth at high concentrations. By
contrast, daidzein, aglycon of puerarin, inhibited growth at three stages of
lettuce protoplast growth and strongly inhibited cell division at 100 μM.
Daidzein might be one cause of the strong inhibitory allelopathic activity of
Kudzu. Grade of inhibitory activities was compared with that of other allelopathic plants including an invader
plant and their allelochemicals studied using the DIA-PP method.