Abstract
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.
Highlights
1.1 Salt Tolerance and Allelopathy of Cultured Mangrove CellsMangrove plants are mainly tree species growing in brackish water varying in salinity in the tropical and subtropical areas (Tomlinson, 1986; Spalding et al, 2010)
3.2 Effects of Four Salts on the Protoplasts Growth of Yellow A. alba Callus In the protoplast cultures of yellow A. alba callus, halophilic nature to NaCl was clearly observed after 12 days of culture, at the cell division stage, when the percentage of divided cells was 43% without additional salts (Figure 2b)
4.1 Protoplast Isolation of Yellow A. alba Callus Protoplasts were isolated in the same enzyme combination, Cellulase RS and Driselase, in 0.6 M mannitol as of the original non-yellow callus of A. alba originating from hypocotyls (Tsuchiya et al, 2013)
Summary
1.1 Salt Tolerance and Allelopathy of Cultured Mangrove CellsMangrove plants are mainly tree species growing in brackish water varying in salinity in the tropical and subtropical areas (Tomlinson, 1986; Spalding et al, 2010). Salt tolerance is a strategy to survive in a high salinity environment. Tissue-cultured cells of several mangrove plants, growing in the coastal region with a high salinity show strong salts tolerance at the cellular level (Kawana & Sasamoto, 2008; Hayashi et al, 2009; Yamamoto et al, 2011). The cellular distribution of salts elements, Na, K, Mg, Ca, Cl, P and S, in suspension-cultured cells of S. alba (Hayatsu et al, 2014) and A. alba (Hayatsu et al, 2017) were investigated using quantitative X-ray microanalysis of cryosections to study the cellular mechanism(s) of strong salt tolerance or halophilism. Allelopathy is a strategy of plants that cannot move away from unfavorable environment, to survive by inhibiting the growth of neighboring plants through production of allelochemicals. Allelopathic activities of many non-mangrove test plants have been studied by the in vitro bioassay method
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