Abstract
protoplasts to examine the allelopathic activities. Protoplasts were isolated with Cellulase R10 and Driselase 20 in 0.6 M mannitol solution and purified by density gradient centrifugation on 0.6 M sucrose. Protoplasts were co-cultured in 50 μL of liquid Murashige and Skoog’s (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid and 0.1 μM benzyladenine and 0.6 M mannitol solution in a 96-well culture plate. Protoplast density ranged from 5 × 103/mL to 105/mL. Cell division of lettuce protoplasts was strongly inhibited by addition of S. ovata protoplasts, and non-spherical cell enlargement was slightly inhibited. By contrast, digital image analysis of scanned 96-well culture platesrevealed no inhibition in accumulation of yellow color in lettuce protoplasts. An anthocyanin, cyanidin 3,5-di-O-glucoside (cyanin), was identified and its content in the red callus was ca. 1 mM of fresh weight. The effects of cyanin on the growth of lettuce protoplasts at three stages were similar to those of red S. ovata protoplasts. From these results, cyanin was most likely the allelochemical contained in red callus of S. ovata. The allelopathic activity of cyanin was compared with that of other putative allelochemicals in several plant materials, using the protoplast co-culture method with digital image analysis.
Highlights
1.1 Salt Tolerance and Allelopathy of Mangrove Cultured CellsMangrove plants are mainly tree species growing in brackish water of different salinity in tropical and subtropical areas (Tomlinson, 1986; Spalding et al, 2010)
Protoplasts isolated from red callus of a mangrove, Sonneratia ovata were co-cultured with recipient lettuce protoplasts to examine the allelopathic activities
Red calluses were induced from all tissues of germinated seedlings, i.e., hypocotyls, cotyledons and roots, of S. ovata (Hasegawa, 2014), and red calluses originating from cotyledons could be sub-cultured for more than four years in the light condition
Summary
Mangrove plants are mainly tree species growing in brackish water of different salinity in tropical and subtropical areas (Tomlinson, 1986; Spalding et al, 2010). Strong salt tolerance at the cellular level of several mangrove plants, growing in the coastal region with a high salinity, has been found using tissue cultured cells and protoplasts (Kawana & Sasamoto, 2008; Hayashi et al, 2009; Yamamoto et al, 2011; Hasegawa et al, 2013). Allelopathic activities of many non-mangrove test plants using lettuce seedlings as recipients have been studied by the in vitro bioassay sandwich method (Fujii, 2000; Takemura et al, 2013). Studies on allelopathy of mangrove plant cells were started only recently, after the in vitro bioassay method of allelopathy using tissue cultured cells and protoplasts was developed
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