Evaluation of Isoflavones as Allelochemicals with Strong Allelopathic Activities of Kudzu Using Protoplast Co-Culture Method with Digital Image Analysis

The inhibitory allelopathic activity of Pueraria montana (Kudzu), and activities of two putative allelochemical isoflavones, puerarin and daidzein, were evaluated using the protoplast co-culture method with digital image analysis using lettuce as a recipient (DIA-PP method). Cotyledon protoplasts of Kudzu were isolated using Cellulase R10 and Driselase 20 in 0.6 M mannitol solution. Optimal hormonal condition and density for growth of Kudzu protoplasts were surveyed. Medium for co-culture of Kudzu or isoflavones with lettuce protoplasts was 50 μl liquid MS basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose, and 0.4 M or 0.6 M mannitol. Protoplast division of lettuce was strongly inhibited by Kudzu at a low density (104/ml). Slightly less inhibition by Kudzu on cell wall formation and yellow pigment accumulation stages of lettuce growth was also observed. Puerarin did not inhibit the growth of lettuce protoplasts at three growth stages but slightly stimulated growth at high concentrations. By contrast, daidzein, aglycon of puerarin, inhibited growth at three stages of lettuce protoplast growth and strongly inhibited cell division at 100 μM. Daidzein might be one cause of the strong inhibitory allelopathic activity of Kudzu. Grade of inhibitory activities was compared with that of other allelopathic plants including an invader plant and their allelochemicals studied using the DIA-PP method.

<i>In Vitro</i>Bioassay of Allelopathy in Four Bamboo Species;<i>Bambusa multiplex, Phyllostachys bambusoides, P. nigra, Sasa kurilensis</i>, Using Sandwich Method and Protoplast Co-Culture Method with Digital Image Analysis

Moderately strong allelopathic activities were found in four bamboo species, Bambusa multiplex cv. Houraichiku;Phyllostachys bambusoides cv. Madake;P. nigra cv. Hachiku;Sasa kurilensis cv. Chishimazasa, which are of different classification or of different ecological distributions, using the “Sandwich Method”, which assays the dried leaves on growth of lettuce seedlings. Only small difference of activity was found among the four bamboo species. In addition, “Protoplast Co-culture Method” for assay of allelopathy in a 50 μL liquid medium using a 96 well culture plate, was applied to the suspension cultures of the four bamboo species. Protoplasts were isolated from two-week cultured suspension cells of four bamboo species using Cellulase RS and Pectolyase Y-23 in 0.6 M mannitol. At low protoplast densities of bamboo, B. multiplex and P. bambusoides stimulated the recipient lettuce growth, i.e., non-spherically cell enlargement and cell divisions observed under an inverted microscope, while protoplasts of P. nigra and S. kurilensis were less stimulatory or inhibitory. Inhibitory effect of S. kurilensis was the strongest among four bamboo species. Furthermore, highly inhibitory effects of S. kurilensis protoplasts on yellow color accumulation of lettuce protoplasts were clearly observed by analysis of a scanned digital image of a 96-well culture plate. Differences and causes of the allelopathic activities were discussed comparing with other plant species studied using the same assay methods.

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

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.

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove Plants, Sonneratia caseolaris and S. alba

The effects of five salt ingredients of sea water, KCl, NaCl, CaCl2, MgCl2 and MgSO4, on induction of cell prolif-eration in Sonneratia caseolaris were investigated. Proliferation was examined in tissue explants derived from such as leaves, cotyledons, and hypocotyls using a small-scale liquid culture method. Addition of 12.5-25 mM of MgCl2 was unique in stimulating cell proliferation in all tissues of S. caseolaris. Otherwise, different effects of salts were observed among the three tissues. In hypocotyl culture, 25-50 mM of NaCl and CaCl2 stimulated cell divisions. Tolerance to 100 mM of MgSO4 was observed in leaves. Three osmotically active compounds commonly used in tissue culture, sorbitol, mannitol and glycinebetaine, were also tested to assess the importance of osmotic effects on cell proliferation. No significant stimulation by these was observed over a wide range of concentrations. Data were compared with those of cotyledon cultures of another mangrove, S. alba, which exhibits no stimulation by MgCl2, stimulation by KCl and tolerance to NaCl. Mechanisms for adaptation of mangrove plants to various and high salts were discussed by comparing the differences in reaction to salts in cultures of two Sonneratia mangrove species of the same genera growing different salt environment.

Relation between Amino Acids Profiles and Recalcitrancy of Cell Growth or Salt Tolerance in Tissue and Protoplast Cultures of Three Mangrove Species, <i>Avicennia alba</i>, <i>Bruguiera sexangula</i>, and <i>Sonneratia alba</i>

Amino acids profiles were investigated in tissues, cultured cells, i.e. callus or suspension cells, and their protoplasts of three mangrove species, Avicennia alba, Bruguiera sexangula, and Sonneratia alba. Original tissues of cultured cells of three mangrove species were cotyledons and hypocotyls, leaves, and cotyledons, respectively. In protoplasts isolated from cultured cells, glutamine and alanine were the major amino acids. Different contents of glycine, proline and serine were observed among protoplasts of three mangrove species. Large differences in the major amino acids were found among cultured cells and their protoplasts while no difference was found between callus and suspension cells independent of additional salt in culture medium. Protoplasts of original tissues, young leaves and cotyledons, contained alanine and glutamine and/or asparagine. In suspension cells of B. sexangula, total contents of amino acids were low while their protoplasts showed similar value as of other samples. Protoplasts of leaf and cotyledons of A. alba and cotyledons of A. lanata, A. marina and S. alba were also investigated. The total contents of amino acids and their profiles might be related to the recalcitrance for the growth and salt tolerance or halophilic nature of cells and basal media used for the maintenance of cell cultures or protoplast cultures of the mangrove species. This is the first report on callus induction from hypocotyls of A. alba.

Strong Allelopathic Activities of Leaves and Cultured Cells of <i>Spiraea thunbergii</i>Assayed by the Protoplast Co-Culture Method with Digital Image Analysis: Evaluation of <i>Cis</i>- and <i>Trans</i>-Cinnamic Acid as Allelochemicals

The inhibitory allelopathic activities of leaves and leaf-origin suspension cultured cells of Spiraea thunbergii, and putative allelochemicals, cis- and trans-cinnamic acid, were investigated by the protoplast co-culture method with digital image analysis (DIA-PP method) using lettuce as a recipient. The optimal conditions of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) for the cell division of S. thunbergii protoplasts were first examined using 50 μL liquid MS basal medium containing 3% sucrose, and 0.8 M mannitol in a 96-well culture plate. The hormonal condition for co-culture, 1 μM 2,4-D plus 0.1 μM BA, which was optimal for lettuce protoplast growth, was sub-optimal for S. thunbergii protoplasts. Effects of co-culture on the three stages of lettuce protoplast growth, i.e., cell wall formation, cell division, and yellow pigment accumulation, were examined. Protoplasts of leaf and suspension cells of S. thunbergii strongly inhibited lettuce protoplast growth at the cell division stage (100% inhibition at 80 - 100 × 103 mL-1), but not so much at the other two stages. Both cis- and trans-cinnamic acid, showed the strongest inhibition at the cell wall formation stage, and 100% inhibition at the cell division stage at 100 μM. These results were compared with those obtained in a lettuce seedling growth test, using different allelopathic plants, and their allelochemicals were studied by the DIA-PP method.