<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

Abstract

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

“…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.…”

“…Recently, the allelopathic activity measured by the sandwich and protoplast co-culture methods has been reported to differ in bamboo species. Some stimulation at low protoplast densities was observed in suspension cell protoplasts of two bamboo species, which showed moderately strong allelopathic activities in the sandwich method [10].…”

“…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.…”

“…In this paper, all protoplast cultures were performed at 28˚C. This temperature was the same as that for bamboo species and co-culture with lettuce [10]. The temperature employed for the protoplast cultures of several mangrove tree species and for the co-culture with lettuce was 30˚C [7].…”

“…Recently, an in vitro bioassay method of allelopathy, the protoplast co-culture method, was developed for herbaceous leguminous plants [6] and for woody or tree plant species using suspension cultured cells and calluses [7] [8] [9] [10]. Effects of the putative allelochemicals and related metabolites were examined using the same lettuce protoplast culture system [9] [11] [12].…”