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 ?M 2,4-dichlorophenoxyacetic acid, 0.1 ?M 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.
Highlights
The tree leguminous plant, Leucaena leucocephala which grows in tropical and subtropical areas, is a highly invasive species
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
Callus of M. pruriens was obtained from protoplast culture of etiolated leaves using Murashige and Skoog (MS) medium with 1 μM 2,4-D and 5 μM BA as described previously (Sasamoto et al, 2013), and sub-cultured on the same medium composition solidified with 0.8% agar under the light condition as lettuce seedlings
Summary
The tree leguminous plant, Leucaena leucocephala which grows in tropical and subtropical areas, is a highly invasive species. Mimosine was found to have an inhibitory effect on influenza viral neuraminidase (Upadhyay et al, 2012) Another woody leguminous plant, Mucuna gigantea that grows in the same (sub) tropical areas, was found to show moderate inhibitory allelopathic activity by the sandwich method (Fujii & Hiradate, 2003). Mucuna gigantea that grows in the same (sub) tropical areas, was found to show moderate inhibitory allelopathic activity by the sandwich method (Fujii & Hiradate, 2003) This species is known to contain high amount of an amino acid, L-DOPA, to another herbaceous M. pruriens. In order to elucidate the cause of the strong allelopathic activities of Leucaena and Mucuna protoplasts, the effect of mimosine in medium on the growth of lettuce protoplasts was investigated and compared with that of L-DOPA (Sasamoto et al, 2013). Functions of two amino acids, mimosine and L-DOPA, and related amino acids as possible allelochemicals in protoplasts of Leucaena and Mucuna are discussed
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