DEVELOPMENTAL STUDIES IN PORPHYRA (RHODOPHYCEAE). III. EFFECT OF CULTURE CONDITIONS ON WALL REGENERATION AND DIFFERENTIATION OF PROTOPLASTS1

Protoplasts isolated from thalli of four Porphyra species regenerated successfully into differentiated plantlets. The efficiency of protoplast isolation and the developmental patterns of the regenerating protoplasts depended on the type of tissues from which they were isolated. However, culture conditions greatly influenced the patterns of development at the cellular and organismal levels. Sorbitol, nitrogen, and agar concentration in the medium controlled rates of cell division, thickening of cell walls, development of rhizoids, and formation of calluses or differentiated blades. Agitation disturbed the attachment of the protoplasts to a substrate. Cells in agitated cultures produced suspensions of single cells and non-polarized small calluses. Calluses which developed from protoplasts survived in storage for over two years. The stored calluses, and cells and protoplasts that were isolated from them, were subcultured successfully. We forsee extensive use of Porphyra cell suspensions for strain selection and vegetative propagation of cultivars. This technology, which makes vegetative cloning of selected Porphyra plants possible, may eliminate the need for cultivation and storage of the conchocelis phase. Protoplasts are also being used as tools for studies in genetic engineering of these commercial species.     

DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal "seed" stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R-10 and 2% Macerozyme R-10. The highest number of protoplasts was obtained on a 50-rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as"seed" stock for algal mass culture.     

SINGLE-CELL PIGMENTATION OF PORPHYRA LINEARIS ANALYZED BY FOURIER TRANSFORM MULTI-PIXEL SPECTROSCOPY AND IMAGE ANALYSIS1

The novel method of Fourier transform multi-pixel spectroscopy was used for the nondestructive analysis of and comparison of pigmentation in different regions of live thalli of the red alga Porphyra linearis. Because the thallus in this alga consists of a monolayer of nonoverlapping cells, we were able to analyze the pigmentation of single cells by combining light absorbance with natural fluorescence data. From the image of each cell in the vegetative male and female reproductive and holdfast regions, more than 4 ± 10⁴ fluorescence and absorbance spectra were obtained. Specific pigments in the different regions were localized by the use of a software program of similarity mapping followed by image construction. The reconstructed images revealed subcellular localization of each pigment according to specific spectroscopic fingerprints. The results showed that the vegetative and female reproductive cell types had a significantly higher content of phycoerythrin than of phycocyanin, and quite similar chlorophyll a levels. Most of the holdfast cells were poorly pigmented, but had more chlorophyll a than phycoerythrin or phycocyanin. The male reproductive cells contained only traces of pigments. Thus, by using Fourier transform multipixel spectroscopy, we were able to characterize the pigmentation of different regions of the thallus and follow the distribution patterns of the different pigments on the subcellular level along the differentiation gradient of the alga.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

Isolation and regeneration of protoplasts from Porphyra dentata and Porphyra crispata

Large numbers of protoplasts (10⁶ to 3·2 × 10⁷ per gram fresh weight) were routinely isolated from two Taiwanese species of Porphyra: P. dentata and P. crispata. Intermediate and final forms of regeneration were observed, including swollen cells, monospore-like cells, filaments, callus-like structures and bladelets. Regeneration of leafy plants was achieved from protoplasts of P. crispata, whereas only conchocelis-like filaments were regenerated from protoplasts of P. dentata. In this latter species, conchosporangia and monosporangia appeared on the filaments upon cultivation at 25 °C. The variability in regenerative patterns is discussed.     

Reproductive biomass allocation in three Sargassum species

Allocation of biomass to sexual reproductive (receptacle) tissue and vegetative (holdfast) tissue differed absolutely and relatively in three Sargassum species that form the bulk of the intertidal algal canopy in the northern Gulf of California. Sargassum herporhizum devoted a greater proportion of its thallus mass into its rhizoidal holdfast than did S. sinicola var. camouii or S. johnstonii, whose holdfasts are solid, more compact, and composed of a lower percentage of water. Conversely, more sexual receptacle tissue was produced by these two species with small holdfasts during the spring reproductive period. Sargassum sinicola var. camouii, which is the only species of the three that becomes fertile in the fall, produces a comparable amount of sexual tissue during this second period of reproduction. Removal of Sargassum from single-species patches showed that canopy regrowth by S. herporhizum with its encroaching rhizoidal holdfast was more rapid and complete than that of the other two species, which invest most of their reproductive efforts into sexual propagules that can disperse long distances. Sargassum herporhizum also displayed a more rapid and complete recovery of canopy cover in patches cleared of thalli and in control patches following the annual summer dieback. These two divergent modes of reproductive biomass allocation suggest that ability to encroach upon nearby open sites and ability to colonize distant discrete islands of suitable habitat represent two distinct reproductive strategies requiring different patterns of biomass allocation. Moreover, for energetic reasons, a species may not be able to excel at both modes of reproduction.     

Regeneration of whole plants from Gracilaria asiatica Chang et Xia protoplasts (Gracilariaceae,Rhodophyta)

A large number of viable protoplasts were produced by enzymatic digestion of Gracilaria asiatica vegetative tissue. The protoplasts underwent initial division after 5–7 d in culture and developed into callus-like cell-masses. Many filaments grew from the periphery of these cell-masses and disappeared after about one month in culture. Simultaneously, the central part of the callus-like cell-masses thickened and its color deepened. The first buds appeared from the center of the cell-masses and developed into whole plants after three months in aerated culture. Many new buds formed around the first plant and more than 20 plants grew per callus-like cell-masses in less than four months. Filaments taken from callus-like cell-masses developed into young plants after about 20 d of culture.     

Evidence for a complex life cycle and endospore formation in the attached, filamentous, segmented bacterium from murine ileum.

Light and electron microscope observations showed that the filamentous, segmented bacterium commonly found attached to the ileal epithelium of rats and mice undergoes a complex life cycle. Filaments comprising up to 90 segments were attached to the microvillous border of absorptive epithelial cells by a specialized terminal holdfast segment. Starting at the free end of the filament and progressing toward the attached end, undifferentiated segments were converted into reproductive or mother segments. Within each mother cell two new holdfast segments developed. As the holdfasts matured, their mother cells degenerated and released them into the intervillar space where they attached, grew, and divided to produce new segmented filaments. Alternately, in some filaments, newly formed but not yet released holdfasts were converted into endospores, which were released in the same manner as holdfasts, presumably to spread the bacterial colony to other members of the rodent population.     

Morphology and reproduction of Bostrychia pinnata (Rhodomelaceae, Ceramiales) in laboratory culture

In unialgal culture, isolates of vegetative plants of Bostrychia pinnata often developed tetrasporangial stichidia and released viable tetraspores. Most tetra‐sporelings developed normal branching before reproduction, however, some sporelings developed procarps, and later, spermatangia on juvenille unbranched stages (< 1 mm). Most normally branched gametophytes (> 2 mm) were initially female before becoming bisexual when older and larger (> 5 mm). Unisexual male gametophytes were not seen in culture. Carposporophytes developed slowly (40–70 days) and were sometimes abortive or produced reduced numbers of carpo‐sporangia (10–15 in cultures compared with 40–50 in field material). Carpospores germinated more slowly than tetraspores and the tetrasporophytes required up to 6 months to reach reproductive maturity. The Polysi‐phonia‐type life‐history was completed in 9–12 months in the laboratory. Peripherohaptera were absent in cultured plants. The isolates from Florida, Guatemala and Peru did not reproduce sexually, but regularly underwent lower branch abscission as a means of vegetative reproduction. After several years in culture, most individual isolates became self‐incompatible. In the female, 1 or 2 procarps formed on each axial segment and most were 4‐celled with a few having 3 cells. A single branched sterile group of 3–7 cells was also present. After fertilization, the diploid nucleus in the carpogonium divided twice, isolating the capping element and trichogyne and establishing the connecting element adjacent to the auxiliary cell. In mature normal cystocarps the sterile group persisted and secreted mucilage into the central cavity. The mature pericarp was four layers thick (one layer of axial filaments and three cortical cell layers). Pseudocystocarps were common, produced no carpo‐sporangia, contained elongate sterile cells, and were enclosed with a partially developed pericarp. Carpogonia in which 4 nuclei were usually seen may indicate developmental failure resulting in pseudocystocarps.     

MOLECULAR ANALYSIS REVEALS CRYPTIC DIVERSITY IN PORPHYRA (RHODOPHYTA)1

The small subunit ribosomal RNA (SSU rRNA) gene was amplified from 15 species of the red alga Porphyra and digested with restriction enzymes to generate data for species identification. The subset of species selected for phylogenetic analysis was P. cuneiforms (Setchell et Hus) Krishnamurthy, P. nereocystis anderson, P. schizophylla Hollenberg et Abbott, P. thuretii Setchell et Dawson and Porphyra 1674. Bangia sp. was used as an out-group. Restriction sites were mapped and used as characters in parsimony and maximum likelihood analysis. The phylogenetic hypotheses generated were compared statistically to possible alternative phylogenies based on traditional morphological taxonomic characters. The results indicate that the current subgenera in Porphyra do not represent monophyletic groups and that traditional morphological and ecological taxonomic characters alone may not be adequate for definitive species identification and cannot be relied on as an indication of Porphyra have large insertions in the SSU gene that are apparently splicesd from the final SSU rRNA molecule. The possible character, distribution and potential significance of these putative introns are discussed.     

VARIATIONS IN THE CELL WALLS AND PHOTOSYNTHETIC PROPERTIES OF PORPHYRA YEZOENSIS (BANGIALES,RHODOPHYTA) DURING ARCHEOSPORE FORMATION1

The formation of archeospores is characteristic of Porphyra yezoensis Ueda and is important for Porphyra aquaculture. Recently, it has been regarded as a valuable seed source for propagation of thalli in mariculture. Cell wall composition changes are associated with archeospore formation in P. yezoensis. Here, we report changes of cell walls of P. yezoensis during archeospore formation. The surfaces of vegetative cells that were originally smooth became rougher and more protuberant as archeosporangia were formed. Ultimately, the cell walls of archeosporangia ruptured, and archeospores were released from the torn cell walls that were left at distal margins of thalli. With changes in cell walls, both effective quantum yield and maximal quantum yield of the same regions in thalli gradually increased during the transformation of vegetative cells to archeospores, suggesting that the photosynthetic properties of the same regions in thalli gradually increased. Meanwhile, photosynthetic parameters for different sectors of thalli were determined, which included the proximal vegetative cells, archeosporangia, and newly released archeospores. The changes in photosynthetic properties of different sectors of thalli were in accordance with that of the same regions in thalli at different stages. In addition, the photosynthetic responses of archeosporangia to light showed higher saturating irradiance levels than those of vegetative cells. All these results suggest that archeosporangial cell walls were not degraded prior to release but were ruptured via bulging of the archeospore within the sporangium, and ultimately, archeospores were discharged. The accumulation of carbohydrates during archeospore formation in P. yezoensis might be required for the release of archeospores.     

Formation and regeneration of protoplasts in the moldRhizopus nigricans

Wall-less protoplasts of the moldRhizopus nigricans were obtained by means of enzymic digestion of the walls of vegetative hyphae by Helix pomatia digestive juice. After removing the enzymes the protoplasts transferred to a nutrition medium regenerate. In the first stage of this process the protoplasts from a firm wall and then the hyphae begin to grow. Regeneration results in the formation of a normal mycelium. If in the course of regeneration of the protoplasts snail enzymes are present, they partially block the synthesis of the wall and the protoplasts transform into incessantly growing giant formations. Re-establishment of morphogenesis begins only after eliminating the effect of the snail enzymes.     

INTERSPECIFIC HYBRIDIZATION IN THE HAPLOID BLADE‐FORMING MARINE CROP PORPHYRA (BANGIALES,RHODOPHYTA): OCCURRENCE OF ALLODIPLOIDY IN SURVIVING F1 GAMETOPHYTIC BLADES1

We performed interspecific hybridization in the haploid blade‐forming marine species (nori) of the genus Porphyra, which have a heteromorphic life cycle with a haploid gametophytic blade and a diploid microscopic sporophyte called the “conchocelis phase.” The green mutant HGT‐6 of P. tenera var. tamatsuensis A. Miura was crossed with the wildtype HG‐1 of P. yezoensis f. narawaensis A. Miura; the F₁ heterozygous conchocelis developed normally and released numerous conchospores. However, almost all the conchospore germlings did not survive past the four‐cell stage or thereabouts, and only a few germlings developed into gametophytic blades. These results indicate that hybrid breakdown occurred during the meiosis, while the surviving F₁ gametophytic blades were considered a breakthrough in the interspecific hybridization of Porphyra. Organelle genomes (cpDNA and mtDNA) were found to be maternally inherited in the interspecific hybridization by molecular analyses of the organelle DNA. In particular, molecular analyses of nuclear DNA revealed that the surviving F₁ blades were allodiploids in the haploid gametophytic phase; however, there is a possibility of the occurrence of rapid chromosomal locus elimination and rearrangement in the F₁ conchocelis phase. Our findings are noteworthy to the breeding of cultivated Porphyra and will provide important information for understanding of the speciation of marine plants with high species diversity.     

Giant kelp vegetative propagation: Adventitious holdfast elements rejuvenate senescent individuals of the Macrocystis pyrifera “integrifolia” ecomorph

Recent findings on holdfast development in the giant kelp highlighted its key importance for Macrocystis vegetative propagation. We report here for the first time the development of adventitious holdfasts from Macrocystis stipes. Swellings emerge spontaneously from different areas of the stipes, especially in senescent or creeping individuals. After being manually fastened to solid substrata, these swellings elongated into haptera, which became strongly attached after 1 month. Within 4 months, new thalli increased in size and vitality, and developed reproductive fronds. Our results suggest the usage of these structures for auxiliary attachment techniques. These could act as a backup, when primary holdfasts are weak, and thus improve the survival rate of the giant kelp in natural beds.     

New life cycles of <Emphasis Type="Italic">Porphyra katadae</Emphasis> var. <Emphasis Type="Italic">hemiphylla</Emphasis> in culture

Porphyra katadae Miura var. hemiphylla Tseng et T. J. Chang, a species distributed around the Liaodong and Shandong Peninsulas of China, produces gametophytes from late winter to early spring. These are monoecious with male and female reproductive tissues in distinct halves or sectors. Vegetative tissues from sectors expected to differentiate into sexual tissue were cultured in the laboratory. Male and female reproductive organs, as well as conchocelis and blades, were differentiated from these tissues. The male and female reproductive tissues were in patches and mixed on the cultured tissue pieces. This was quite different from the wild-type sectored individuals. The F₁ conchospore germlings also produced monospores, carposporangia, spermatangia and conchocelis. These carposporangia and spermatangia were in patches and were mixed on the F₁ fronds. The results imply that P. katadae var. hemiphylla is possibly sex-differentiated rather than sex-determined. This is the first report of such a dimorphic life history in the genus Porphyra.     

MEIOSIS,BLADE DEVELOPMENT,AND SEX DETERMINATION IN PORPHYRA PURPUREA (RHODOPHYTA)1

The discovery in the early 1980s that meiosis occurs during germination of conchospores of Porphyra yezoensis Ueda suggested that the sexually divided fronds of Porphyra purpurea (Roth) C. Agardh might similarly originate from meiotic segregation of a pair of sex-determining alleles during early sporeling development. After establishing conditions suitable for propagating P. purpurea in culture, observations on developing sporelings demonstrated that meiosis takes place during the first two divisions of the germinating conchospores. In the first division, the spore is split into an upper and lower cell. In the second, an anticlinal division in the upper cell yields two daughter cells situated one beside the other, and a periclinal division in the bottom cell gives two cells arranged one above the other. Thus, during normal development, the first four cells of the sporeling constitute a meiotic tetrad whose cells are arranged in a characteristic fashion. Stable color mutants of P. purpurea were isolated, genetically characterized, and used as genetic markers to follow the fate of individual cells of the tetrad during subsequent frond development. Nearly the entire blade of the mature thallus is derived from the two upper cells of the tetrad, with the two lower cells mostly giving rise to the rhizoidal holdfast region. Cell lineage boundaries laid down by the segregation of color alleles at meiosis corresponded perfectly with those later defined by sexual differentiation on the same fronds, strongly supporting the hypothesis that sex determination in P. purpurea is controlled by alleles at a segregating chromosomal locus.     

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-μm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal. Received: 21 April 1997 / Revision received: 27 June 1997 / Accepted: 5 July 1997     

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.