Protoplast Co-culture Bioassay for Allelopathy in Leguminous Plants, Leucaena leucocephala and Mucuna gigantea, Containing Allelochemical Amino Acids, Mimosine and L-DOPA

Abstract

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.

Evaluation of Allelochemicals, Abscisic Acid and Coumarin, in Leaf-Origin Suspension Cultured Cells of <i>Prunus yedoensis</i> Using Protoplast Co-Culture Bioassay Method

“…This value is similar to that of Sasa kurilensis, which showed the strongest inhibition among the four bamboo species [10], and is between the Derris indica [8] and the invader plant, Leucaena leucocephala protoplasts, which totally inhibited the lettuce protoplast growth at 10 4 /mL [9]. Protoplasts of peduncles-origin P. yedoensis and P. lannesiana were less inhibitory than leaf-origin P. yedoensis.…”

“…Compared with the previous data of many plant species using the sandwich method [2] [18], Prunus in this report is in the group of very strong allelopathic activities, though young leaves of Prunus were used in this report. Stronger activity was also obtained in the younger leaves of Mucuna gigantea [9]. With the sandwich method, bacterial or fungal contamination may occur during the 3 days of incubation.…”

“…Compared with the inhibitory effects of leaves of several plant species investigated using the sandwich method (Derris indica [8]; Mucuna gigantea, Leucaena leucocephala [9], Bamboo species [10]), more than 90% inhibition by 50 mg leaves is very strong. Especially, no growth of lettuce root obtained with leaves of P. yedoensis was the strongest of all.…”

“…Especially, no growth of lettuce root obtained with leaves of P. yedoensis was the strongest of all. Leaves of an invader plant, Leucaena leucocephala, inhibited the root growth by 90% in the sandwich method [9].…”

“…Effects of the putative allelochemicals and related metabolites were examined using the same lettuce protoplast culture system [9] [11] [12]. In this report, we first examined the allelopathic activities of young leaves of P. yedoensis and P. lannesiana using the sandwich method.…”