To study allelopathy at the cellular level, we established a new bioassay method using protoplasts from etiolated leaves of a leguminous plant, Mucuna pruriens , which contains a large amount of L-3,4-dihydroxyphenylalanine (L-DOPA) and protoplasts prepared from cotyledons, leaves or roots of Lactuca sativa (lettuce) grown in an axenic condition. The bioassay was performed using 96 multi-well culture plates and required only a small volume of medium, 50 ?L, per well. For the mixed culture of Mucuna and recipient lettuce protoplast, the optimum condition was Murashige and Skoog’s basal medium, containing 1 ?M of 2,4-dichlorophenoxyacetic acid, 5 ?M of benzyladenine and 0.4 M mannitol solution. The inhibitory effect of M. pruriens protoplasts was found to vary with the density of the recipient L. sativa protoplasts. The M. pruriens protoplasts had no effect on the division of protoplasts from suspension cells of Oryza sativa . These results were in agreement with those obtained by the conventional plant box method. Our study offered a novel in vitro assay method, which will be widely applicable to study allelopathy of various plants on lettuce and also for quantitative studies between plant species under different culture conditions in order to simulate the possible future environmental risk.

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 μm, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for β-1,4-glucan and β-1,3-glucan.

We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.

Respiration and related aspects of metabolism were investigated in the roots and leaves of 2-year-old trees of the mangrove plant, Avicennia marina in the presence of 100, 250 and 500 mᴍ NaCl. The rate of respiration of leaves increased with increasing concentrations of NaCl in the incubation medium, but respiration of roots was not similarly affected. In order to examine the relative rates of catabolism of glucose by the glycolysis-tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate pathway (PP pathway), we determined the rates of release of ¹⁴CO₂ from [1-¹⁴C]glucose and from [ 6 -¹⁴C]glucose in segments of roots and leaves. The ratios of rates (C₆/C₁) in roots varied from 0.30 to 0.44, while ratios of 0.85 to 0.99 were obtained when leaves were incubated in the presence of various concentrations of NaCl. It appeared that the PP pathway was more involved in sugar catabolism in the roots than in the leaves of A. marina. Uniformaly ¹⁴C-labelled sucrose, incubated with segments of roots and leaves for 18 h, was converted to CO₂, amino acids (mainly glutamine), organic acids (mainly malic acid), sugars and ethanol-insoluble macromolecules. The incorporation of radioactivity into most of these components was not significantly affected by NaCl. However, in leaves (but not in roots) the release of ¹⁴CO₂ from [ U -¹⁴C]sucrose was en­ hanced by NaCl at 250 mᴍ and 500 mᴍ, while the rate of incorporation of radioactivity into macromolecules was reduced by high concentrations of NaCl. Incorporation of radioactivity from [ U -¹⁴C]sucrose into malic acid was enhanced in both roots and leaves by an increase in the concentration of NaCl from 100 mᴍ to 500 mᴍ (this concentrations is similar to that in sea water). Independent of the concentration of NaCl, more than half of the radioactivity in the neutral fraction from leaves was incorporated into an unidentified sugar, while in the same fraction from roots, the radioactivity was associated with glucose, fructose and sucrose. On the basis of these results, a discussion is presented of the characteristics of catabolism of sugars in A. marina in relation to salt resistance.