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

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.

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

“…Image analysis of yellow pigment accumulation of lettuce protoplasts was performed after 3 weeks to 1.5 months of culture as described previously [17] [22] [23] [24] [25] [27] [29] [31]. Digital image of a 96-well culture plate was scanned using a scanner (Epson GTX-970).…”

“…Strong inhibition of yellow pigment accumulation by Sasa kurilensis was calculated as in line b2 in Figure 5(b), which shows strong inhibition at the cell wall formation stage similar to Kudzu. Such a calculation method (Subtraction of the control yellow values without lettuce protoplasts, at each density of test plant protoplasts or at each concentration of allelochemicals) had been employed in the previously reported papers using the DIA-PP method [17] [22]- [27] [29] [31].…”

“…A carotenoid was extracted to hexane fraction from lettuce protoplasts that had accumulated the yellow pigment [22]. Recently, a very different pattern of inhibition at three growth stages of lettuce by a carotenoid-accumulating callus of Avicennia alba has been reported [27]. Very recently, unique allelopathic activities of three carotenoids were investigated at three growth stages of lettuce protoplast using the DIA-PP method [34].…”

“…The digital image analysis with protoplast co-culture method (DIA-PP method), which is the analysis of scanned image of yellow pigment accumulation in co-cultured (with lettuce protoplast) 96-well plate, was further developed. Numbers of stu-died plants and their putative allelochemicals are increasing as follows: Arabidopsis thaliana [22] [23]; four bamboo species [24]; Sonneratia ovata (anthocyanin [25]); Spiraea thunbergii (cinnamic acid) and S. cantoniensis (anthocyanin) [26]; halophilic mangrove plant, Avicennia alba (carotenoid) [27]; Coffea canephora (caffeine and metabolites) [28] [29] [30]; V. villosa (canavanine and cyanamide) [17]. Very recently, volatile compounds of Saffron (safranal) and Spiraea thunbergii (tulipalin A) were reported [31].…”

Comparision of several secondary metabolite and elemental ion contents of leaves from <i> Kandelia obovata </i> and <i> Sonneratia caseolaris </i> forests located in the Red River delta

“…A significant reduction was observed in lutein content of pumpkin leaves when exposed to ultra violet light (Norshazila et al, 2017). A recent study reported that the protoplast cultures of yellow A. alba callus, which contained carotenoids, were halophilic to NaCl, KCl, and MgCl 2 but not to CaCl 2 (Sasamoto et al, 2020). Banerjee (2017) revealed that in mangrove plants exposed to salinity, carotenoids were significantly reduced.…”