Evaluation of an Anthocyanin, Cyanidin 3,5-di-O-glucoside, as an Allelochemical in Red Callus of a Mangrove Sonneratia ovata, Using Protoplast Co-Culture Bioassay Method with Digital Image Analysis

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.

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Allelopathy in a Leguminous Mangrove Plant, Derris indica: Protoplast Co-culture Bioassay and Rotenone Effect

Natural Product Communications ◽

10.1177/1934578x1501000512 ◽

2015 ◽

Vol 10 (5) ◽

pp. 1934578X1501000 ◽

Cited By ~ 2

Author(s):

Aya Inoue ◽

Daisuke Mori ◽

Reiko Minagawa ◽

Yoshiharu Fujii ◽

Hamako Sasamoto

Keyword(s):

Suspension Culture ◽

Basal Medium ◽

Culture Method ◽

Mangrove Plant ◽

Lettuce Seedling ◽

Mannitol Solution ◽

Allelopathic Activity ◽

Young Leaves ◽

2,4 Dichlorophenoxyacetic Acid ◽

Derris Indica

To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the ‘Protoplasts Co-culture Method’ for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 μM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 μM 2,4-D and 0.1 μM BA. The protoplast density of D. indica used in co-culturing varied from 6 × 103 - 105 / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica.

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High Allelopathic Activity of Carotenoid-accumulating Callus of a Halophilic Mangrove Plant, Avicennia alba: Protoplast Co-culture Method with Digital Image Analysis

Journal of Plant Studies ◽

10.5539/jps.v9n1p1 ◽

2019 ◽

Vol 9 (1) ◽

pp. 1

Author(s):

Hamako Sasamoto ◽

Manabu Hayatsu ◽

Suechika Suzuki

Keyword(s):

Image Analysis ◽

Digital Image ◽

Digital Image Analysis ◽

Culture Method ◽

Yellow Pigment ◽

Growth Stages ◽

Salt Tolerant ◽

Mangrove Plant ◽

Allelopathic Activity ◽

Avicennia Alba

A yellow callus strain was established from hypocotyls of a halophilic mangrove plant, Avicennia alba, by subculture in the dark. Allelopathic activities of yellow A. alba callus were assayed using recipient lettuce protoplasts at three growth stages by the protoplast co-culture method with digital image analysis. The protoplast cultures of yellow A. alba callus were halophilic to NaCl, KCl, and MgCl2 (up to 200 mM) but not to CaCl2. By contrast, NaCl and KCl inhibited the growth of non-salt-tolerant lettuce protoplasts, while CaCl2 and MgCl2 stimulated their growth at low concentrations. Highly salt-tolerant or halophilic mangrove plant cells were expected to have low allelopathic activity, but the protoplasts of yellow A. alba callus had very strong allelopathic activity. The inhibition was strongest at the cell division stage with growth being inhibited to 50% and 9% of the control by 104 mL-1 and 5 × 104 mL-1 of A. alba, respectively. There was less inhibition at the yellow pigment accumulation stage of lettuce. Stimulation was observed at the early cell wall formation stage with up to 105 mL-1 of A. alba. The yellow pigment of yellow A. alba callus was extracted with hexane and its absorption spectrum showed the wavelength peaks of a carotenoid, neoxanthin. Using transmission electron microscopy, specific electron-dense structures were found in yellow A. alba callus, which were similar to the undeveloped ultrastructure of a carotenoid. A carotenoid was strongly suggested to be the putative allelochemical in yellow callus of A. alba.

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