Amino acids profiles were investigated in tissues, cultured cells, i.e. callus or suspension cells, and their protoplasts of three mangrove species, Avicennia alba, Bruguiera sexangula, and Sonneratia alba. Original tissues of cultured cells of three mangrove species were cotyledons and hypocotyls, leaves, and cotyledons, respectively. In protoplasts isolated from cultured cells, glutamine and alanine were the major amino acids. Different contents of glycine, proline and serine were observed among protoplasts of three mangrove species. Large differences in the major amino acids were found among cultured cells and their protoplasts while no difference was found between callus and suspension cells independent of additional salt in culture medium. Protoplasts of original tissues, young leaves and cotyledons, contained alanine and glutamine and/or asparagine. In suspension cells of B. sexangula, total contents of amino acids were low while their protoplasts showed similar value as of other samples. Protoplasts of leaf and cotyledons of A. alba and cotyledons of A. lanata, A. marina and S. alba were also investigated. The total contents of amino acids and their profiles might be related to the recalcitrance for the growth and salt tolerance or halophilic nature of cells and basal media used for the maintenance of cell cultures or protoplast cultures of the mangrove species. This is the first report on callus induction from hypocotyls of A. alba.
Two cultured cell lines were developed from cotyledons of a halophyte mangrove, Avicennia alba. In the high-Ca callus line, which was sub-cultured in a modified amino acid medium containing 3 mM CaCl 2 , growth of calluses and their protoplasts were both inhibited by low concentrations of CaCl 2 in the culture medium. Removal of Ca 2+ from the culture medium stimulated callus growth and the calluses could be sub-cultured without CaCl 2 (low-Ca callus line). The intra-(cytoplasmic matrix and vacuole) and extra-(cell wall) cellular concentrations of elements, i.e., [Ca] [S] were investigated using quantitative X-ray microanalysis of cryosections of calluses from both cell lines. [Ca] was high in the cytoplasmic matrix and cell wall of the high-Ca line. [Ca] was lowered in the low-Ca line in all cell compartments, though still detected. Ca-containing electron-dense precipitates were accumulated in the middle lamella of cell walls in resin-embedded sections of the high-Ca line. CaCl 2 in the medium stimulated protoplast growth only in the low-Ca line. These results suggested that a low cellular [Ca] is needed for protoplasts growth of A. alba. The importance of cellular [Ca] for the growth of halophilic mangrove plant cells was discussed.
Callus induction, maintenance and protoplast cultures were achieved from immature seeds of a woody leguminous mangrove, Caesalpinia crista. Axenic cultures were possible during 1.5 months of pod storage in 0.1% benzalkonium chloride solution. Callus induction was achieved using 1 mL liquid medium in a 10 mL flat-bottomed culture tube. Protoplasts were isolated using Cellulase R10, Hemicellulase, and Driselase 20 in 0.6 M mannitol solution and sub-culturable calluses were obtained in 50 μL liquid medium using a 96-microplate method. The optimal hormonal concentration was 10 μM each of 2,4-dichlorophenoxyacetic acid and benzyladenine in liquid Murashige and Skoog's basal medium for both callus induction and maintenance, and protoplast cultures. Similarities and differences in amino acid profiles and culture conditions are discussed among woody mangrove species and non-mangrove leguminous species. Caesalpinia crista cultures were unique as they secreted a large amount of amino acids, including proline, into the liquid culture medium.
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