In vitro Bioassay of Allelopathic Activities of a Mangrove Tree, Kandelia obovata, and Fast-growing Trees, Betula platyphylla and Populus alba, Using Protoplast Co-culture Method

Allelopathic activities of a salt-tolerant and low-temperature tolerant mangrove tree, Kandelia obovata, which grows in brackish water regions of sub-tropical areas, and two fast-growing trees, Betula platyphylla and Populus alba, which grow in the temperate area, were examined by two in vitro bioassay methods, the sandwich method using dried leaves and the protoplast co-culture method using leaf protoplasts. Lettuce root growth examined by the sandwich method, was inhibited 50% by 50 mg dried mature leaves of K. obovata. In the protoplast co-culture method, inhibition rates of cell division of lettuce protoplasts were 31% and 69% by leaf protoplasts of K. obovata at densities of 1 × 104 mL-1 and 5 × 104 mL-1, respectively. These results were compared with the inverse relationship between allelopathic activities and salt tolerance of mangrove plants of different families. B. platyphylla showed 37% inhibition by the sandwich method using dried young leaves, but only 10% inhibition at 5 × 104 mL-1 by the protoplast co-culture method using leaf protoplasts of B. platyphylla. Dried young leaves of P. alba showed 66% inhibition, but the leaf protoplasts at the density of 5 × 104 mL-1 showed highly stimulatory activity. Abscisic acid, of which contents in leaf protoplasts of three tree species varies from high to low in relation to salt tolerance and recalcitrance of tissue culture, was discussed as a putative allelochemical.

Protoplast-Based Transient Expression Combined with Plant Cultivation Systems as a Valuable Tool for Floral Thermogenesis Studies in Aroids

Floral thermogenesis in plants plays a significant role in their reproductive function. Thermogenic aroids constitute a large family in highly thermogenic angiosperms, many of which possess intense heat-producing abilities. Several genes have been proposed to be involved in floral thermogenesis of aroids, but the biological tools to identify the functions of those genes at cellular and molecular levels are lacking. Among the many thermogenic aroids, we focused on skunk cabbage (Symplocarpus renifolius) because of its ability to produce intense, durable heat and small aboveground parts compared with other thermogenic aroids. In this study, leaf protoplasts were isolated from potted and shoot tip-cultured skunk cabbage plants and used to develop transient assay systems. The isolation protocol included an additional, sucrose gradient centrifugation step, which yielded high-purity protoplasts from both types of plants. The isolation and transfection efficiency of the protoplasts exceeded 1.0 × 105/g fresh weight and 50%, respectively, in both potted and shoot tip-cultured plants. Using this protoplast-based transient expression (PTE) system, we determined the protein localization of three mitochondrial energy-dissipating proteins, SrAOX, SrUCPA, and SrNDA1, fused to green fluorescent protein (GFP). In skunk cabbage leaf protoplasts, these three GFP-fused proteins were localized in MitoTracker-stained mitochondria. However, the green fluorescent particles in protoplasts expressing SrUCPA-GFP were enlarged compared with those in protoplasts expressing SrAOX-GFP and SrNDA1-GFP. Our PTE system is a powerful tool for functional gene analysis not only in thermogenic aroids but also in non-thermogenic aroids.