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

Abstract

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 x 10 3-10 5 / 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.

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

“…Strong inhibition was observed at the cell division stage of lettuce protoplast growth after 12 days of co-culture. There was less than 50% and 10% growth of control at 10 4 mL -1 and at 5 × 10 4 mL -1 , respectively, with A. alba, which was similar to the findings reported for upstream grown mangrove species, e.g., Sonneratia caseolaris (Hasegawa et al, 2014) and Derris indica (Inoue et al, 2015). The mangrove plant, S. ovata grows in-between of seaward-side grown S. alba and S. caseolaris.…”

“…An inverse relationship had been found between allelopathic activity and salt tolerance at both the plant level and cellular level in three Sonneratia mangrove species which can grow in most seaward-side (S. alba) and upstream areas (S. caseolaris) and in-between (S. ovata) using dark-grown suspension-cultured cells (Hasegawa et al, 2014). High allelopathic activities have been found using both the sandwich method and protoplast co-culture method in a leguminous tree mangrove, Derris indica, which grows in the upstream area (Inoue et al, 2015). Such an inverse relationship between salt tolerance and allelopathic activities might be widely applicable to different mangrove plant species (Hasegawa, 2014;.…”

“…The inhibitory effects of putative allelochemicals in plants, e.g., L-DOPA in Mucuna (Sasamoto et al, 2013;Mori et al, 2015); purine alkaloids, caffeine metabolites (Sasamoto et al, 2015a); pyridine alkaloids, mimosine in L. leucocephala (Mori et al, 2015); nicotinic acid, nicotinamide and trigonelline (Sasamoto & Ashihara, 2014); abscisic acid and coumarin in Prunus (Fujise et al, 2018); canavanine and cyanamide in Vicia (Sasamoto et al, 2019) have been determined using the same lettuce protoplast co-culture method. However, mangrove cultured cells have not been examined for putative allelochemicals, except for an isoflavonoid, rotenone, in D. indica (Inoue et al, 2015). Very recently, a natural pigment, an anthocyanin, cyanidin 3,5-di-O-glucoside (cyanin), was identified in red callus of S. ovata originated from cotyledons, which was long sub-cultured in the light condition.…”

A Protocol for Axenic Liquid Cell Cultures of a Woody Leguminous Mangrove, Caesalpinia crista, and their Amino Acids Profiling

“…In the non-mangrove woody legume, Leucaena leucocephala, very high concentrations of either BA or a strong cytokinin, TDZ, were needed for callus and protoplast cultures [24]. In the tree legume mangrove, Derris indica, a similar tendency was observed [13].…”

“…Other than the species from the Rhizophoraceae, i.e. Bruguiera sexangula [14], callus regeneration from protoplast culture of mangrove species has proven to be difficult, even after initial successful cell divisions [13,[15][16][17]. The relationship between amino acid profiles and optimal basal media, i.e., Murashige and Skoog's (MS) [18] and the modified amino acid (mAA) [11][12]17], was investigated in three mangrove families, Sonneratiaceae, Rhizophoraceae, and Avicenniaceae [6], but not in the Leguminosae.…”