Protoplasts isolated from calluses of two woody leguminous plants, Leucaena leucocephala and Mucuna gigantea, which contain high levels of the allelochemical amino acids, mimosine and L-DOPA, respectively, were co-cultured to examine their allelopathic activities. The young leaves were first confirmed to have strong inhibitory activity by the sandwich method. Callus cultures were induced and sub-cultured from seeds of L. leucocephala and from leaves of M. gigantea in Murashige and Skoog's (MS) basal media containing high concentrations of auxin and cytokinins. Protoplasts of both calluses were isolated using the separate well method under strong enzymatic conditions, in high osmotic, 0.8 M mannitol solution. Protoplasts of both species had strong inhibitory effects on the recipient protoplasts of cotyledons of Lactuca sativa (lettuce) co-cultured using 96 multi-well culture plates in the MS basal medium, containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine and 0.8 M mannitol solution. Mimosine was less inhibitory than L-DOPA on the growth of lettuce protoplasts at the stage of colony formation. Amino acid profiles of calluses and protoplasts of the above two species and Mucuna pruriens showed very low contents of mimosine and L-DOPA, respectively. The cause of the high inhibitory allelopathic activities found in Leucaena and Mucuna protoplasts on the growth of lettuce protoplasts was discussed.
Dried leaves of Prunus yedoensis and P. lannesiana (50 mg) showed strong inhibitory allelopathic activities, e.g., more than 97% growth inhibition of lettuce seedling using the sandwich method. Similarly, among suspension cultures induced from leaves and peduncles of two Prunus species, we found the strongest inhibitory allelopathic activities of protoplasts of leaf-origin suspension cells of P. yedoensis, when the protoplast co-culture method for bioassay of allelopathy was applied with lettuce as a recipient plant. Effects of two putative allelochemicals, abscisic acid and coumarin, on both protoplast cultures of lettuce and P. yedoensis were investigated. Coumarin inhibited the growth of lettuce protoplasts from low concentrations, while abscisic acid stimulated.Abscisic acid inhibited the protoplast growth of P. yedoensis from low concentrations, while coumarin did not, but inhibited only at a high concentration (1 mM). Contents of abscisic acid in protoplasts were measured using small scale purification and Enzyme Linked Immno Sorbent Assay, and contents of coumarin in leaf-origin susepension cells of P. yedoensis were measured using Gas Chromatography-Mass Spectrometry. Coumarin was more likely the allelochemical causing the strong inhibitory allelopathic activities of P. yedoensis in the protoplast co-culture bioassay. Effectiveness of the protoplast co-culture bioassay method of allelopathy was discussed.
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