Donald P. Cheney

Phenolic-nitrogen interactions in eelgrass, Zostera marina L.: possible implications for disease resistance

Abstract Experiments with eelgrass, Zostera marina L., transplanted into running seawater mesocosms with either a mud or sand substratum indicated that leaf nitrogen and phenolic contents were inversely related. A caffeic acid ester accounted for ∼ 50% of the phenolic content; sulfated flavones made up most of the remainder. Plants with a low phenolic content grown in mud suffered complete mortality from wasting disease, while plants with a high phenolic content in sand were still growing well when the experiment was terminated.

Protoplast isolation and cell division in the agar-producing seaweed Gracilaria (Rhodophyta)

Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber‐van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid‐producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R‐10, 3% Macerozyme R‐10, 1% agarase and 0.5% Pectolyase Y‐ 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2‐6 days; cell division in 5‐10 days. Protoplast‐produced cell masses up to the 16‐32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.

Some effects of plant growth regulators on tissue cultures of the marine red alga Agardhiella subulata (Gigartinales, Rhodophyta)

We examined whether auxins and cytokinins, either singly or in combination, stimulate cell division in tissue cultures of a red seaweed. Our experimental model consisted of filamentous and callus-like growths that developed from cross-sectional discs cut from young branches of Agardhiella subulata. Plant growth regulators were added to the medium to give combinations of an auxin with a cytokinin over a range of concentrations (1 µg L−1 −10 mg L−1). Several mixtures of auxins and cytokinins, as well as some single auxins, cytokinins and phenolics, stimulated cell division and growth in the tissue cultures beyond that of controls. The treatments that were effective included: phenylacetic acid/zeatin; phenylacetic acid/6-benzylaminopurine; α-naphthaleneacetic acid/zeatin; 2,4,5-trichlorophenoxyacetic acid/6-benzylaminopurine; and indoleacetic acid/kinetin. High concentrations of cytokinins (i.e. 10 mg L−1) inhibited the regeneration of plants in some of the cell cultures. These results provide further evidence that growth regulators can be used for the tissue culture of seaweeds and for the study of developmental phenomena in these plants.

Discrimination of Porphyra species based on small subunit ribosomal RNA gene sequence

The complete nucleotide sequences of ssu rRNA genes were determined for nine species of Porphyra. Ssu rRNA gene structure was classified into four types by the presence and absence of intron(s). Gene structure even differed within the same species. Exon nucleotide sequences were identical within the same species, but differed among species. Seventeen species of Porphyra were discriminated by comparing the sequences of these diversified regions, using the results of this study and previous studies.

Carrageenan analysis of tissue cultures and whole plants of Agardhiella subulata

For the past several years, our laboratory has been involved in developing basic protoplast and tissue culture techniques for carrageenan- and agar-producing seaweeds which can potentially be used for strain improvement. Both types of techniques offer new opportunities for genetic improvement in seaweeds not available using classical plant breeding methods (Cheney, 1986). While protoplast and tissue culture techniques are well developed and widely applied to higher plants today for crop improvement, their application to seaweeds is just beginning.

Isolation of humic acid from the brown algaPilayella littoralis

A standard humic acid extraction procedure has been used to isolate dark brown organic residues from samples of the macroscopic marine brown algaPilayella littoralis. The residues are insoluble in water, but soluble at high pH, and are similar in elemental composition, ash content, UV-visible, IR, PMR and X-Ray fluorescence spectra, X-Ray diffractograms and scanning electron micrographs to residues of a humic acid isolated from municipal compost. These results indicate thatPilayella produces humic acids.

The isolation and characterization of agardhilactone, a novel oxylipin from the marine red alga Agardhiella subulata

Abstract A novel tricyclic oxylipin containing δ-lactone, cyclopentane and epoxide rings and a conjugated diene, has been isolated from the marine red alga Agardhiella subulata. The structure, including partial stereochemistry, was determined by NMR and GC-MS analysis of menthoxycarbonyl derivatives.

COMPARISON OF DEVELOPMENT AND PHOTOSYNTHETIC GROWTH FOR FILAMENT CLUMPS AND REGENERATED MICROPLANTLET CULTURES OF AGARDHIELLA SUBULATA (RHODOPHYTA, GIGARTINALES)

Two axenic, in vitro liquid suspension cultures were established for Agardhiella subulata (C. Agardh) Kraft et Wynne, and their growth characteristics were compared. This study illustrated how reliable routes for the development of suspension cultures of macrophytic red algae of terete thallus morphology can be achieved for biotechnology applications. Undifferentiated filament clumps of 2–8 mm diameter were established by induction of callus‐like tissue from thallus explants, and lightly branched microplantlets of 2–10 mm length were established by regeneration of filament clumps. The filament clumps were susceptible to regeneration. Adventitious shoot formation was reliably induced from 40% to 70% of the filament clumps by gentle mixing at 100 rev min−1 on an orbital shaker. The specific growth rate of the microplantlets was higher than the filament clumps in nonagitated well plate culture (4%–6% per day for microplantlets vs. 2%–3% per day for filament clumps) at 24° C and 8–36 μmol photons·m−2·s−1 irradiance (10:14 h LD cycle) when grown on ASP12 artificial seawater medium at pH 8.6–8.9 with 20%–25% per day medium replacement. Oxygen evolution rate vs. irradiance measurements showed that relative to the filament clumps, microplantlets had a higher maximum specific oxygen evolution rate (Po,max= 0.181 ± 0.035 vs. 0.130 ± 0.023 mmol O2·g−1 dry cell mass·h−1), but comparable respiration rate (Qo= 0.040 ± 0.013 vs. 0.033 ± 0.017 mmol O2·g−1 dry cell mass·h−1), compensation point (Ic= 3.8 ± 2.4 vs. 5.7 ± 1.2 μmol photons·m−2·s−1), and light intensity at 63.2% of saturation (Ik= 17.5 ± 3.9 vs. 14.9 ± 2.6 μmol photons·m−2·s−1). The microplantlet culture was more suitable for suspension culture development than the filament clump culture because it was morphologically stable and exhibited higher growth rates.

One step antibiotic disk method for obtaining axenic cultures of multicellular marine algae

A simple and rapid method for obtaining axenic plant material has been devised where antibiotic-containing paper disks are applied to nutrient agarose or agar plates upon which small pieces of plant have been spread. By applying multiple disks to the agarose plate, susceptibility to a large number of antibiotics can be tested simultaneously. Clear zones are produced around those disks where contaminating bacteria are susceptible. Plant pieces are then removed from the clear zones and separately tested for sterility to identify the axenic pieces. The method has been successfully applied to multicellular marine algae (e.g.Enteromorpha, Porphyra, laminaria, Gracilaria andAgardhiella). Pieces fromAgardhiella plants survive better on nutrient medium solidified with agarose when α-naphthaleneacetic acid and zeatin are present in the medium.

HALOGENATED MONOTERPENE PRODUCTION IN REGENERATED PLANTLET CULTURES OF OCHTODES SECUNDIRAMEA (RHODOPHYTA, CRYPTONEMIALES)

Three genera of macrophytic red algae (Ochtodes, Plocamium, and Portieria) contain novel halogenated monoterpenes. To develop an in vitro system for studying halogenated monoterpene production, a laboratory tissue culture was established for Ochtodes secundiramea (Montagne) Howe (Cryptonemiales, Rhizophyllidaceae). Specifically, callus cells were induced from thallus explants of O. secundiramea plants. Shoot primordia regenerated from callus cells and developed into plantlets that released tetraspores. A sporeling from one of these tetraspores was selected for further culture. Axenic plantlets were cultivated in ESS‐enriched natural seawater. Thallus tissue was cut into small pieces before subculture. Each plantlet grew as a symmetrical array of highly branched shoot tissues emanating from a common center, ultimately assuming a spherical shape of 20 mm diameter 4 weeks after subculture. Specific growth rates of over 20% per day were attained in bubble‐aerated flask culture at an optimal temperature of 26° C and photosynthetic saturation light intensity of 200 μmol photons·m−2·s−1. The cultured plantlets contained myrcene and seven halogenated monoterpenes, based on gas chromatography–mass spectroscopy analysis of dichloromethane extracts. Although bromomyrcene was the dominant acyclic halogenated monoterpene, the cyclic halogenated monoterpenes chondrocole C and ochtodene were also produced by the O. secundiramea plantlet cultures. Halogenated monoterpene production at light‐saturated growth conditions increased with decreasing nitrogen availability below 1.0 mM medium nitrate concentration (N:P ratios of 1.6:1 to 32:1). The halogenated monoterpene yield was insensitive to medium nitrate concentrations above 1.0 mM (N:P ratios of 32:1 to 320:1), where the bromomyrcene yield was 1700 μg per gram of dry cell mass.