Conchospore Germination Research Articles

Effects of high temperature stress on conchospore germination and early seedling development ofPyropia haitanensis

Pyropia haitanensis is one of the main cultivated macroalgae in China. In recent years, the cultivation of P. haitanensis conchospores in seawater is conducted in advance to obtain earlier harvest. However, the high seawater temperature may exert negative impacts on the algae. Seven temperature conditions (25, 26, 27, 28, 29, 30 and 31°C) were set in the culture of P. haitanensis to investigate the effects of temperature on the conchospore germination and early seedling development of P. haitanensis. The results showed that conchospore germination rate decreased with the increase in temperature. The temperatures of 25 and 26°C were suitable for P. haitanensis conchospore germination. Higher temperature (27°C) promoted conchospore germination on the 1st and 2nd days but decreased the germination considerably on the 3rd day. The conchospore mortality rate reached 89% after culture at high temperature (31°C) for 4 days. Additionally, the development of conchospore seedlings was normal, the total number of cells increased, and the survival rate was high (53%–90%) in the range of 25–29°C. At 25°C, the absolute growth rate of seedlings was as high as 302%. However, the development slowed down and the absolute growth rate decreased with the increase in temperature. At 30 and 31°C, seedling development was abnormal or even ceased, the survival rate was as low as 20% (30°C) and 11% (31°C), and the absolute growth rates were negative under the two conditions. The mortality of seedling was remarkably higher at 31°C than at other temperatures and gradually increased with the increase in temperature (25–31°C). And, the potential mechanisms underlying high temperature damages were analysed. Therefore, the temperature range suitable for the seedling collection and cultivation of P. haitanensis conchospore is 25–29°C.

Effects of short‐wavelength ultraviolet ray irradiation on the early development and induction of pigmentation mutants of conchospore germlings in Pyropia haitanensis (Rhodophyta)

Ultraviolet mutagenesis is a safe and efficient method to induce mutations for breeding new germplasm of laver. In this study, high-frequency short-wave ultraviolet (UV-C) irradiation (λ = 254 nm) with different doses (50, 100, 200, 300, 400 and 500 J m−2) was used to induce colour mutants during the germination of conchospores in Pyropia haitanensis. The early development of conchospore germination and the morphogenesis of tetrad germlings were observed, which revealed that low-dose irradiation (50 J m−2) promoted the germination and early development of conchospores, while irradiation doses above 100 J m−2 inhibited the germination of conchospores and growth of tetrad germlings. In the dose range of 50–500 J m−2, the survival rate and germination rate of conchospores gradually decreased, while the mutation rate of colour mutants increased first and then decreased with increasing doses. The highest mutation rate was observed with the dose of 300 J m−2 or with irradiation time 72 s. These results indicated that the optimal UV-C irradiation dose was 300 J m−2 for the mutagenesis of P. haitanensis conchospores. In addition, UV-C irradiation also had a significant effect on the early development of conchospores and phenotypes of pigmentation mutant arranged in tetrad germlings, which was mainly manifested in the large number of colour-sectored blades produced by irradiated conchospores and in the decrease in their aspect ratios. This study provides new insights in carrying out UV-C mutagenesis for the breeding of P. haitanensis.

Thallus sectioning as an efficient monospore release method in Pyropia yezoensis (Bangiales, Rhodophyta)

The red seaweed Pyropia yezoensis is a marine crop of important economic value and is widely cultivated in the coastal areas of northern China. The current seedling-raising system relies on the germination of conchospores, a process not fast enough to meet the increasing demand from farmers. In this study, we developed a monospore-dependent seedling method based on the asexual reproduction of the Pyropia thallus. The Pyropia thallus was physically sectioned into small pieces (microthalli) that were cultivated at 15 °C. The algal cells in the microthalli became morphologically condensed, underwent cell division, and then developed into monospores on the 5th day. The monospores were able to attach to the seeding rope in 24 h and germinated into healthy thalli. To optimize the efficiency of monospore release, we tested the effect of temperature and the size and original positions of the microthalli as well as the age of the mother thallus. Microthalli with a size of 30–50 cells from the middle and apex of 21-day-old thalli cultivated at 15 °C yielded the optimum production of monospores. Theoretically, in this thallus-to-thallus seeding strategy, 0.1 g of thallus could produce at least 107 monospores, with the same number of offspring thallus seedlings. Taking all of these advantages together, including the high efficiency, short time, low cost and easy operation, this physical sectioning method could serve as a promising seed source especially for new cultivars with superior traits in Pyropia farming and rapid replenishment of seedlings when thalli undergo disastrous diseases.

Cultivation of early life history stages of Porphyra dioica from the British Isles

Bladed Bangiales of the genus Porphyra/Pyropia are highly valuable red algae and extensively farmed in South East Asia. Interest is rising in cultivating species local to the North East Atlantic but the control of the heteromorphic life cycle of native species remains difficult as previous studies reported high inter- and intraspecific variability in required cultivation conditions. Here, working with Porphyra dioica from a UK source population, we conducted a series of experiments investigating the influence of substrate, temperature, photoperiod and light intensity on the development of early life history stages (conchocelis (filamentous sporophyte) and young thalli (gametophyte)). Special focus was the influence of temperature and photoperiod on mature conchocelis to induce a conchospore mass release—the current bottleneck of European Porphyra cultivation. Sporophytes grew largest on an oyster shell substrate and under long day conditions at 18 °C. A decrease in temperature from 18 to 9 °C initiated a mass conchospore release (498 ± 146 spores mL−1) from a P. dioica conchocelis culture grown in suspension. Released conchospores germinated into small thalli on nylon ropes, with best growth (7.2 ± 0.9% day−1) at low temperatures of 9 °C. Conchospore germination increased with decreasing light intensity but germination success was generally very low (< 5%), indicating the cultivation protocol needs further improvement. Our results reflect the adaptation of P. diocia to seasonal environmental conditions in temperate regions and the importance of these conditions for the successful cultivation. We are the first to describe a mass conchospore release for P. diocia growing in suspension which has important implications for commercial production.

Morphogenesis and spontaneous chromosome doubling during the parthenogenetic development of haploid female gametophytes in Pyropia haitanensis (Bangiales, Rhodophyta)

Pyropia haitanensis is one of the most economically important seaweeds growing in the coastal regions of south China. Previous studies revealed that parthenogenesis could emerge in P. haitanensis under certain culture conditions. In the present study, detailed cytological observations were made of morphogenesis and chromosomal behaviors of female gametophytes of P. haitanensis during parthenogenesis. Unisexual cultures of female gametophytes, derived from isolated somatic cells of wild gametophytes or obtained from cloned sporophytes, ultimately led to their development into parthenosporophytes. Based on the changes in shape and color of the cellular chromatophore, as well as the metamorphosis and condition of the released cells, the parthenogenesis of P. haitanensis was divided into six developmental stages: vegetative cells (VCs) with multiple stellate chromatophores, carpogonium-like cells (CLCs) with dispersed chromatophores, carpogonium-like protoplasts (CLPs) released post-cell wall dissolution, carpospore-like cells (CCs) with regenerated cell wall, carpospore-like germlings (CGs), and parthenosporophytes. Chromosomal observations showed that VCs were haploid (n = 5), small proportions of CLCs, and CLPs and CCs were diploid (2n = 10), while all the CGs, filamentous conchocelis cells, and conchosporangial branch cells of parthenosporophytes were diploid (2n = 10). Haploid, aneuploid, and polyploidy were not observed in the latter group of cells. Spontaneous chromosome doubling occurred during the parthenogenesis of P. haitanensis, first in highly differentiated CLCs. Furthermore, pit connections could be seen throughout the parthenosporophyte development process similar to that in heterozygous sporophytes. The chromosomes were seen undergoing meiosis during the germination of conchospores released from mature parthenosporophytes, leading to the generation of tetrad cells, which developed into haploid female gametophytes. These results provide evidence for better understanding the mechanism underlying parthenogenesis in Pyropia.

Study of mosaicism of Pyropia yezoensis

ABSTRACTCytological observations and sequence analysis of the ribosomal intergenic spacer (IGS) were conducted on conchospores and thalli of Pyropia yezoensis from China. The complete progress of conchospore germination of P. yezoensis was observed, and a tracking study of P. yezoensis mosaics was conducted by microscope, our results showed that the thallus is haploid, with three chromosomes; and the conchocelis is diploid, with six chromosomes. Conchospore germination of P. yezoensis developed in a linear pattern, and cell divisions were not synchronous. The thallus of P. yezoensis was divided into ten parts in our study, IGS was used as a molecular marker to conduct a sequence analysis of different portions of a complete thallus (the reproductive region, nutrient region, and base). P. yezoensis is mosaic of different genetic material based on molecular level differences among the IGS sequences of the different parts of P. yezoensis. The thallus was homozygous; with either two or three mosaics. Chimerism of Pyropia was proven at both the morphological and the molecular level. The results of our study supported the dominant view that meiosis of Pyropia occurs during conchospore germination.

Isolation and characterization of an improved strain of Porphyra chauhanii (Bangiales, Rhodophyta) with high-temperature resistance

The blades of the wild-type strain of Porphyra chauhanii (PC-WT) were irradiated with 60Co-γ rays and cultured at high temperature (30 °C) for 25 days. The surviving somatic cells of the irradiated blades were isolated enzymatically and were regenerated into whole blades and screened at high temperature. Vigorously growing blades at high temperature were selected and individually proliferated. A genetically homozygous and free-living conchocelis strain of TR-5 was obtained through parthenogenesis. Subsequently, TR-5 was compared with PC-WT for high-temperature resistance, growth, contents of major photosynthetic pigments, and the releasing ability of monospores and conchospores. The results showed that the survival percentage, division, and rhizoid germination of conchospores were not significantly different between TR-5 and PC-WT at 18 and 23 °C; but the survival percentages were 261.2 and 329.3 % higher, the division percentages were 42.8 and 72.6 % higher, and the rhizoid germination percentages were 94.3 and 199.5 % higher in the former than in the latter at 27 and 29 °C, respectively. When F1 gametophytic blades of TR-5 and PC-WT were firstly cultured at 23 °C for 30 days, and then cultured at 18, 23, and 27 °C for another 10 days, the absolute growth rates of TR-5 blades were 4.4, 4.3, and 10.4 times those of PC-WT and the relative growth rates were 1.1, 1.1, and 2.0 times those of PC-WT, respectively. The apical and middle parts of PC-WT blades were disintegrated due to the release of a large number of monospores after culturing for 15 days at 27 °C or 10 days at 29 °C, and only the basal parts of the blades remained intact. On the other hand, TR-5 blades grew rapidly with normal color and shape without releasing monospores and were slightly curled, even after being cultured for an additional 30 days. In addition, the contents of three major photosynthetic pigments (chlorophyll a (Chl. a), phycoerythrin (PE), and phycocyanin (PC)) and the conchospore numbers in TR-5 at a suitable temperature (23 °C) were 66.7, 244.2, 115.7, and 92.0 % higher than those of PC-WT, respectively. Meanwhile, the blades of TR-5 were 23.2 % thinner than those of PC-WT. The results indicated that TR-5 has thinner blade, higher contents of the major photosynthetic pigments, faster growth speed, higher temperature resistance, has larger number of conchospores released, and no monospore release as compared with PC-WT. It therefore has potential to be developed into a new variety suitable for cultivation in the aquaculture areas with high temperature in the coast of South China.

Early development patterns and morphogenesis of blades in four species of Porphyra (Bangiales, Rhodophyta)

Pigment mutants were used as genetic markers to study the early development and morphogenesis of blades in four species of Porphyra. In Porphyra haitanensis, P. yezoensis, and P. oligospermatangia, the first two divisions are transverse during conchospore germination, yielding four cells arranged in a line. These species are representative of linear development pattern in Porphyra. Resulting in blades with color sectors vertically arranged. In P. katadai var. hemiphylla, the first division is transverse and the upper cell divides vertically forming two side-by-side cells, and its blades are derived mostly from the upper cell showing a bilateral development pattern with two lateral parts of different colors. In this type of germination, most or the entire blade is derived from the upper cells. Some fronds of P. katadai var. hemiphylla developed in linear pattern. In addition, 9.3% of the conchospore germlings of linear development were produced at 10°C, 15.3% at 15°C, and 38.0% at 20°C for conchospore germlings of P. katadai var. hemiphylla. More linear development occurred at higher temperatures. The results revealed general trends of early developmental patterns and morphogenesis of blades within the genus of Porphyra. Developmental patterns and morphogenesis of blades are under the influence of temperatures.

Observations on the division characterization of diploid nuclear in Porphyra (Bangiales, Rhodophyta)

Nuclear divisions of carpospores, conchocelis and conchospores of Porphyra yezoensis, P. haitanensis, P. katadai var. hemiphylla and P. oligospermatangia from China were investigated. The observations showed diploid chromosome numbers of 2n = 6 for P. yezoensis and P. oligospermatangia, and 2n = 10 for P. haitanensis and P. katadai var. hemiphylla. For all four species, somatic pairing of chromosome sets was observed in late prophase. Sister chromosomes separated at anaphase as mitosis took place in carpospores, conchocelis filamentous cells, conchosporangial branch cells and sporangial cells (conchospore formation). Chromosome configurations of tetrad and ring-shaped in conchospore germination were observed, demonstrating the occurrence of meiosis. The characteristics of diploid nuclear division in 2n = 6 species are the same as those of 2n = 10 species. The influence of somatic pairing on nuclear division of diploid cells in Porphyra was discussed.

Identification of Porphyra yezoensis (Rhodophyta) meiosis by DNA quantification using confocal laser scanning microscopy

Conchospore germlings of Porphyra yezoensis were stained with a fluorescent dye for DNA and observed with confocal laser scanning microscopy (CLSM). Relative DNA values of the germling nuclei were obtained by measuring fluorescence intensities of nuclear regions of the optically sliced specimens, using the mean value of the smallest blade cells as a reference of the genomic n value. Such quantification revealed that the nuclear DNA amounts of the one-cell, two-cell, and four-cell-stage germlings are approximately 4 × n, 2 × n, and n ∼2 × n values respectively; these values agreed well with the expected ones from the hypothesis that meiosis corresponds to the first successive cell divisions after the conchospore germination. These results are consistent with a previous study on cytogenetic analysis of the chimaera blade formation (Ohme and Miura 1988, Plant Sci 57:135–140) and not consistent with a recent microscopic study (Wang et al. 2006, Phycol Res 54:201–207) which proposed that the first meiotic division occurs at the conchospore formation and the second division at the germination.

Effects of solar UV radiation on germination of conchospores and morphogenesis of sporelings in Porphyra haitanensis (Rhodophyta)

The effects of ultraviolet radiation (UVR 280-400 nm) on the germination of Porphyra haitanensis conchospores and on the growth and morphogenesis of the subsequent sporelings were investigated by culturing the released conchospores under natural sunlight from 29 September to 6 October 2005. Germination increased with time and was faster when UV-B was excluded using cut-off filters. There were significant negative effects of UV-B radiation on growth and cell division of sporelings, with decreases up to 18% for thallus length, between 6 and 18% for thallus width, up to 29% for thallus area, and between 6 and 14% for cell size as compared to PAR-controls. UV-A had a significant positive effect on morphogenesis, enhancing the formation of sporelings with cells dividing transversely; on the other hand, UV-B delayed the formation of such sporelings. We also tested the effects of solar UVR on the growth of P. haitanensis juveniles and found no significant effects. Our results indicate that UV-A has an important role in the germination and morphogenesis of the species, but on the other hand, sporelings of P. haitanensis are more sensitive to UV-B radiation than juveniles.

Nuclear division of the vegetative cells, conchosporangial cells and conchospores of Porphyra yezoensis (Bangiales, Rhodophyta)

SUMMARYTo confirm the position and timing of meiosis in Porphyra yezoensis Ueda, the nuclear division of vegetative cells, conchosporangial cells and conchospores was observed. An improved staining method using modified carbol fuchsin was introduced to stain the chromosomes of Porphyra. Pit‐connections between conchosporangial cells also stained well with this method. Leptotene, zygotene, pachytene, diplotene, diakinesis, metaphase, anaphase and telophase were observed in the conchosporangial cells. During the germination of conchospores, no characteristics of meiosis I were found. No difference between the nuclear division of vegetative cells and that of conchospores was observed, and 2–3 days were needed for the first cell division both in vegetative cells and conchospores. Therefore, the cell division that occurs during conchospore germination is not meiosis I. Our results indicate that the prophase of meiosis I begins during the formation of conchosporangial branches, and metaphase I, anaphase I and telophase I take place during the maturation of conchosporangial branches. Then the three‐bivalent nucleate sporangia complete cell division to form two individual conchospores, each with one three‐univalent nucleus. The conchospores released from the sporangia are at meiotic interphase. Meiosis II occurs at the first nuclear division during conchospore germination, which is a possible explanation for the observation of mosaic thalli in mutant germlings of P. yezoensis. The mosaic thalli might also arise from gene conversion/post meiotic segregation events, comparable to those in Sordaria fimicola (Roberge ex Desm.) Ces. &amp; De Not. and Neurospora crassa Shear &amp; B.O. Dodge.

DEVELOPMENT OF THE CONCHOCELIS STAGE OF PORPHYRA PERFORATA FROM THE TEMPERATE MEXICAN PACIFIC

Salgado‐Rogel M. L2,3 &amp; Zertuche‐González J. A.1 1Instituto de Investigaciones Oceanológicas UABC, Ensenada B.C. PO BOX 453 México 22860; 2Instituto Nacional de la Pesca, Centro Regional de Investigaciones Pesqueras, Ensenada, B.C. PO BOX 1306 México 22860. 3Facultad de Ciencias Marinas UABC, Ensenada B.C. PO BOX 453 México 22860.The conditions governing the reproduction of the conchocelis stage of Porphyra perforata were studied in vitro. Carpospores and conchocelis development were observed under a factorial of light and temperature with four levels of irradiance 15, 50, 100 and 275μEm‐2s‐1, with 8:16,12:12,16:8 photoperiods and four temperature levels 10,15, 20 y 25 °C. Conchocelis stage and conchosporangial structure developed without shells. Filaments were present at 10‐20°C and 15‐270μEm‐2s‐1 with 8:16, 12:12 ,16:8 photoperiods. The initiation of the conchosporangia was present at 50‐100μEm‐2s‐1. Release and germination of conchospores occurred only when the photoperiod changed from 8: 16 to 12:12. Filaments exposed to 270μEm‐2s‐1 (16:8) persisted with discolored apices that did not develop conchosporangia. Filaments not exposed to variations of photoperiod formed monospores. Our results are notoriously different from previous studies of P. perforata from Washington USA (cold region) were Conchocelis was vegetative at 15°C and 5‐100μEm‐2s‐1, and concho‐sporangial initiation was occurred at 10‐25μEm‐2s‐1 with a 8:16 photoperiod and only in shells as substrate. Differences were found between both regions in the levels of temperature, irradiance and photoperiod, which promoted the development of the reproductive structure of the Conchocelis stage. These results show that the conchocelis stage in plants from the temperate region develop and produce reproductive structures at higher light and temperature than plants from the cold region, which suggests ecophysiological differences of this species between both regions.

海藻（スサビノリ殻胞子）を用いた生物検定による都市下水の塩素代替消毒処理水の毒性比較

Chlorination of secondary effluents produce strong toxic substance such as monochloramine or oxidant on growth of aquatic organisms. Therefore, alternative disinfection methods to chlorination should be considered. However, the effect of effluents disinfected with the alternatives on seaweed has not been clarified. In this study, the toxicity of secondary effluents disinfected with chlorine (NaOCl), chlorine dioxide (ClO2), ozone (O3), and ultraviolet rays (UV) were investigated by bioassay using Porphyra yezoensis conchospores. Secondary effluents used in this study were disinfected with disinfectants or rays which inactivated coliform groups in secondary effluents of 99.9%. Lowest-observed-effect concentrations (LOECs) of secondary effluents disinfected with NaOCl, ClO2, and O3 for germination ratio of conchospores were 1.0, 10, and 100% of effluent concentration, respectively. The toxicity of effluents disinfected with NaOCl and ClO2 were very strong. In contrast, the effluents disinfected with UV rays did not affect the germination of conchospores. The toxicity of effluents disinfected with NaOCl, ClO2, and O3 were considered to depend on initial total residual oxidant (TRO). Dechlorination with Na2SO3 of effluents disinfected with NaOCl or ClO2 did not affect the germination of conchospores, even in 100% of effluent concentration.

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

ABSTRACTThe 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.

Photoperiod and temperature regulation of the life history of Porphyra columbina (Rhodophyta, Bangiales) from central Chile

The effects of temperature (10, 15, 20 °C) and photoperiod (8:16, 12:12, 16:8 (hours of light: hours of dark)) on the growth and reproduction of different stages in the life history of Porphyra columbina Montagne were assessed. The conchocelis phase had higher growth rates at 12 h light:12 h dark, 45 μmol m−2 s−1, and 15 °C. Under all the temperatures and photoperiods tested, the conchocelis phase could propagate either by monospores or vegetative fragmentation. Conchosporangia were formed only at 10 °C and 8 h light:16 h dark or at 15 °C and either 8 h light:16 h dark or 12 h light:12 h dark. Long photoperiods inhibited conchosporangia formation at all temperatures tested with the exception of 20 °C and 16 h light:8 h dark, where sporangia was formed once. Conchospores were released when the temperature was decreased (15 to 10 °C) maintaining the photoperiod. Conchospore germination occurred under the three photoperiods and temperatures tested. Growth of juvenile fronds was maximum at 15 °C and either 12 h light:12 h dark or 16 h light:8 h dark. Fronds formed carpospores and spermatia at 15 °C and 16 h light:8 h dark only. With optimum conditions the life history was completed in 60 days. Spermatia had three chromosomes (n = 3).