‘Seed’ production of Porphyra spp. by tissue culture

Differentiation of archeospores was observed from excised tissue of young thalli of various monoecious Porphyra species ( P. tenera, P. yezoensis, P. suborbiculata, P. okamurae) after 4–8 days in culture at temperatures of 20 and 25 °C. Excised tissue from adult thalli did not differentiate into archeospores, but rather regenerated directly into blades and rhizoids of foliose thalli. Tissues from young thalli of two dioecious Porphyra species ( P. dentata and P. pseudolinearis) also regenerated into blades and rhizoids after manipulation of the culture conditions. In addition, 1–2 celled tissue pieces of both monoecious and dioecious species were also seen to develop directly into blades. Polarity of regeneration of blades and rhizoids was observed in these species. These results suggest that ‘seed’ can be obtained through tissue culture instead of using conventional conchocelis culture for commercial nori aquaculture in Japan. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular divergence of the ssu rRNA gene and internal transcribed spacer 1 in Porphyra yezoensis (Rhodophyta)

Nucleotide sequences from the downstream of ssu rDNA to ITS1 region of the individual thalli of both wild-collected Porphyra yezoensis from three different sites and culture strains were determined to obtain the molecular features of strains in the P. yezoensis lineage. Wild-collected thalli identified by morphological systematics, included the individuals that were separate from the P. yezoensis lineage based on ssu rDNA and ITS1 sequence homologies and phylogenetic relationships constructed using ITS1 sequences. Ssu rDNA exon region nucleotide sequences were identical among the wild-collected and clture strains of P. yezoensis. However, all individual wild-collected P. yezoensis thalli had different ITS1 sequences, even among individuals from the same sites. Furthermore, two different ssu rDNA structures with and without an intron were found in individuals from the same site. These results indicated the possibility that the presence and sequence of introns and ITS1 sequences can be used as a characteristic to determine the origin of culture strains. Four of six culture strains examined had an identical sequence from the ssu rDNA to ITS1, while the sequences of another two strains differed. In this study, wild-collected and culture strain thalli sequences were not identical, although similar pairs were identified. This revised version was published online in July 2006 with corrections to the Cover Date.     

THE LIFE CYCLE OE SARGASSUM HORNERI (PHAEOPHYTA) IN LABORATORY CULTURE1

The life cycle of the large dioecious alga Sargassum horneri (Turner) C. Agardh was completed in unialgal culture by controlling photoperiod in relation to the phase of growth. Embryos isolated from a naturally grown female thallus gave rise to early germlings that rapidly formed blades under both short-day (9 h L) and long-day (15 h L) conditions at 20° C Shoot elongation, which followed early blade formation, occurred under the short-day conditions hut not under the long-day conditions. Functional female and male receptacles developed when thalli 8–14 cm long grown under the short-day conditions were transferred to the long-day conditions; gamete fusion occurred when male and female thalli were grown together. Fertilized oospores gave rise to normal thalli in a manner similar to that for in situ plants. Thus, the life cycle of S. horneri was completed in laboratory culture.     

ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN AXENIC CULTURE AND ITS RESPONSE TO THE ENDOPHYTIC FUNGUS MYCOSPHAERELLA ASCOPHYLLI AND EPIPHYTIC BACTERIA1

Ascophyllum nodosum (L.) Le Jol. was freed from bacteria and the endophytic fungus Mycosphaerella ascophylli Cotton by repeated treatment with chlorine solutions and grown in artificial seawater. Two types of axenic culture of different origin were obtained. Type 1 was developed from apices of A. nodosum collected in the sea. Type 2 was from plants which developed from adventitious embryos on rhizoids formed by type 1. This is the first time A. nodosum has been cultivated axenically. Growth of the axenic alga was increased by IAA, 21P and zeatin. Without external growth regulators some strains of the axenic alga deteriorated within a year; others developed a filamentous habit. Sulfur in a reduced state also stimulated growth. Addition of either glucose, mannose or mannitol to the medium caused the formation of calluslike layers of loosely packed colorless cells under the epidermis of the thalli and the epidermis was sloughed off. No increase in thallus length was noticed. Mycosphaerella ascophylli in axenic culture did not excude any substances stimulating growth of the alga, but that does not exclude an influence of the fungus on the alga in vivo. The fungus, when growing within the alga, seemed to have little influence on algal morphology. A bacterized but fungus-free A. nodosum was cultivated in an artificial seawater for 8 years. In the bacteria-free alga, the fungus protruded from the epidermis and evidently utilized the alga as a carbon source. The bacteria thus seem much more important than the fungus for normal growth of the Ascophyllum plant.     

高盐下条斑紫菜光合特性和S-腺苷甲硫氨酸合成酶基因表达的变化

为了研究条斑紫菜耐盐机理,对条斑紫菜叶状体进行了高盐胁迫处理,继而采用氧电极法测量了光合放氧速率和呼吸耗氧速率的变化,采用实时荧光定量PCR技术测量了S-腺苷甲硫氨酸合成酶(命名为PySAMS)基因的表达变化。结果显示藻体的光合与呼吸作用均受到高盐度海水的显著影响,随着盐度的增加,光合放氧率逐渐降低,呼吸耗氧率也逐渐降低。高盐度海水对PySAMS基因表达量也产生了显著影响,40和50盐度的海水诱导了PySAMS表达,但60至80盐度的海水却不同程度地抑制了PySAMS表达。据此推测,在面对较高盐度胁迫时条斑紫菜叶状体将逐步降低体内新陈代谢以度过不良环境。     

On the Differentiation and Development of Cells Isolated from Thalli of Porphyra yezoensis Ueda

Cells were isolated after enzyme treatment from the thalli of Porphyra yezoensis Ueda and cultured in enriched seawater media (MES). Cultures were put in an incubator with light intensity of 2000--2500 lx from cool-white flurescent lights and were maintained on a 12:12 LD cycle. The temperature was 20±2℃ and the media were changed weekly. Two main types of differentiation and development ways in cultured cells under light microscope: 1. Cells directly formed the single cell seedlings Eke monospores; 2. Cells first divided into cell aggregates, then released lots of spores that could germinate into sporelings. Both single cell seedlings and sporelings were called cell seedlings. So it is possibole to breed from the ceils isolated directly from the thalli in Porphyra yezoensis.     

Isolation of porphyran-degrading marine microorganisms from the surface of red alga, Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.     

坛紫菜叶状体的无菌化培养及其应用

对坛紫菜叶状体的无菌处理方法进行了优化,并利用紫菜外生菌对无菌处理后的紫菜叶片进行了人工感染。经0.7%KI和0.1%(w/v)氨苄青霉素对紫菜叶片进行预处理后,利用氨苄青霉素、硫酸链霉素、新霉素、庆大霉素及卡那霉素等5种抗生素及其不同组合对紫菜叶片进行处理,筛选出最佳抗生素组合、浓度和培养条件。结果表明:氨苄青霉素(终浓度300μg/mL)、卡那霉素(终浓度100μg/mL)与庆大霉素(终浓度100μg/mL)3种抗生素组合对坛紫菜叶状体进行无菌处理18 h,对紫菜细胞的毒害较小,并且3种抗生素合用对89.5%紫菜外生细菌的抑菌率达80%以上;外生菌感染结果研究表明:用105/mL真菌孢子液和细菌菌液回染紫菜,培养22 d后,实验组紫菜生长状况较对照组好,对照组紫菜出现褪色。用108/mL真菌孢子液分别回染健康紫菜、穿刺紫菜(用灭菌刀片在紫菜表面划1～2 mm的伤口),分别在18℃、28℃和35℃条件下培养。实验结果表明28℃和35℃高温和穿刺均会影响无菌紫菜健康生长,且加菌紫菜在高温和穿刺条件下,则会出现明显病斑,而18℃培养的加菌紫菜则生长良好。     

The life history of Porphyra endiviifolium from the South Shetland Islands, Antarctica

This paper describes the reproduction and life history of an intertidal species, Porphyra endiviifolium, from Antarctica. Field specimens were examined microscopically, prepared for electron microscopy and used to establish cultures. Wild populations comprised two kinds of leafy thalli, morphologically similar but distinguished by their mode of reproduction, either sexual or asexual. Carpospores from monoecious leafy gametophytes developed into conchocelis filaments in culture, and under “winter-spring” conditions these formed conchospores that germinated to produce leafy thalli. Monospores from asexual leafy thalli developed directly into two different forms of leafy thalli. Only one of the cultured morphotypes became fertile, reproducing asexually by monospores. We conclude that the phases of the life history of P. endiviifolium show different ecological strategies, the conchocelis phase reproducing in response to short days unlike the leafy thalli in which growth and reproduction respond primarily to irradiance. Received: 2 May 1997 / Accepted: 11 October 1997     

Isolation of Hyphomonas Strains that Induce Normal Morphogenesis in Protoplasts of the Marine Red Alga Pyropia yezoensis

Marine macroalgae cannot develop normal morphology under axenic conditions although normal morphogenesis can be sustained when certain bacteria are present. In this study, bacteria that induced normal morphogenesis in the red alga Pyropia yezoensis (Nori) were identified. The bacteria were isolated from algal media, thalli, tissue debris, and purified protoplasts during protoplast isolation from P. yezoensis laboratory cultures. 16S rRNA gene sequence analysis showed these bacterial isolates belonged to α-Proteobacteria (12 groups), γ-Proteobacteria (3 groups), and Flavobacteria (2 groups). Axenic protoplasts of P. yezoensis generated by removing epiphytic bacteria were co-cultured along with the bacterial isolates. Most axenic protoplasts showed irregular morphogenetic and anaplastic cells; cells with normal morphology were scarce. However, inoculation with 11 strains of Hyphomonas (α-Proteobacteria) led to significantly higher normal morphogenetic rates (4.5–7.3 %, P?Hyphomonas strains were recovered from all experiments; thus, certain Hyphomonas strains can induce normal morphogenesis in P. yezoensis protoplasts. Direct inoculation of the Hyphomonas strain exhibited higher morphogenetic activity than inoculation of its extracellular and intracellular products. This is the first study demonstrating the influence of specific bacteria on protoplast morphology in marine macroalgae.     

Porphyra lilliputiana sp. nov. (Bangiales, Rhodophyta): A diminutive New Zealand endemic with novel reproductive biology

A new species of Porphyra, Porphyra lilliputiana, is described for the New Zealand region. This species is very small ([5] 10–20 [35] mm) and is found growing epiphytically, epilithically and epizoically on upper inter-tidal shores of moderate exposure. Field-collected material of P. lilliputiana possessed archeosporangia, endosporangia, spermatangia and zygotosporangia. In culture, archeospores vi/ere released and germinated to form thalli. Endosporangia either developed directly into thalli or released endospores which individually formed thalli. Zygotospores developed into the concho-celis phase, which formed conchosporangia. Released conchospores formed thalli. This species is distinguished by its small size, arrangement of reproductive cells, occurrence of endosporangia, dentate margin and habitat.     

Molecular analysis of physiological responses to changes in nitrogen in a marine macroalga, Porphyra yezoensis (Rhodophyta)

The rhodophyte seaweed Porphyra yezoensis, known more commonly world-wide as "nori", is an important commercial crop in Japan. Cultivation of nori in Japan is often affected by outbreaks of "iroochi", a discoloration of the thalli due to a decrease in inorganic nutrients in the culture area that in turn decreases the amount of photosynthetic pigments in the thalli. Treating thalli with inorganic nitrogen can reverse iroochi. In this paper, we report on the characterization of three P. yezoensis genes, a nitrate transporter (PyNRT2) and two urea transporters (PyUT1 and PyUT2), which may be involved in reversing iroochi. The predicted length of the PyNRT2 protein was 479 amino acids (AA), while PyUT1 and PyUT2 were 740 and 680 AA, respectively. PyNRT2 was more similar to NRT2 from a chromophyte than to NRTs from Chlamydomonas and higher plants. The two P. yezoensis UTs had 56% AA identity to each other, and showed the closest relationship to higher plant and yeast DUR3 proteins which formed a subfamily of the sodium-solute symporter protein family. Hydrophobicity plots of the AA sequences showed that the PyNRT2, PyUT1, and PyUT2 included 12, 15, and 16 transmembrane domains, respectively. Southern blot analysis indicated that the P. yezoensis genome has a single NRT2-encoding gene and at least four UT-encoding genes. Expression analysis of PyNRT2 and PyUT genes showed that the messenger RNA level of the PyNRT2 gene reached a maximum after 48 h in the nitrate starvation condition and was then restored to the constitutive level, while expression of the PyUT genes was induced in proportion to treatment times in the nitrate starvation condition. These results suggest that the PyNRT2 and PyUT are responsible for the high-affinity nitrate/urea transport systems that operate under low external nitrate concentrations.     

A novel technique for propagation of Porphyra yezoensis Ueda blades in suspension cultures via monospores

The present methods for propagation of Porphyra blades in suspension cultures are inadequate for commercial production. A novel method for propagation of P. yezoensis blades in suspension cultures was tested. Blades were asexually propagated via monospores in suspension by cutting blades of various sizes into differently sized tissue sections and inoculating these in fresh medium. After 2–3 weeks, the sections completely disintegrated, producing monospores followed by a dense suspension culture of blades. This technique shows great promise for producing multiple crops from one initial blade inoculum, increasing production, and simplifying the propagation of this non-fragmenting alga in land-based tank mariculture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of DNA preparation from laver (Porphyra yezoensis) thalli on reproducibility of RAPD (random amplified polymorphic DNA) patterns

Laver (Porphyra yezoensis) DNAs were extracted from thalli with five different procedures and used for RAPD (random amplified polymorphic DNA) analysis as templates. Restriction enzyme-digestive DNAs were obtained with all procedures examined. However, RAPD patterns generated with these DNAs appeared highly irreproducible and were considerably different from each other. When DNAs purified with CsCl gradient centrifugation were used for RAPD analysis as templates, highly reproducible RAPD patterns were obtained, suggesting that unpurified DNAs extracted from thalli with all five extraction procedures contained an excess of RNA, polysaccharides and/or other materials which affected the RAPD reproducibility. Thus, results indicated that purification of DNA is essential to produce reproducible RAPD patterns of Porphyra DNA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Porphyra cell cultures: isolation,growth and polysaccharide production

A range of cell lines was isolated fromPorphyra umbilicalis L. (Rhodophyta) tissue using a variety of methods, the most successful involving exposure to a limpet acetone powder enzyme extract for 24 h, homogenisation and filtration through a series of polyester meshes. All established lines grew as 0.1–5 mm diameter aggregates in liquid culture; most were stable and have been grown in shake-flask or air-lift culture for periods in excess of 1 yr without reverting to the foliose growth form. An investigation of the medium used to grow these lines indicated that it was not nitrogen-deficient and that the sodium chloride concentration was optimal. The addition of an organic buffer increased the final cell yield. None of these cell lines grew heterotrophically in medium supplemented with a range of fixed carbon sources. The infrared spectra of polysaccharides isolated fromPorphyra aggregates and from tissue grown under identical conditions indicated that the structures of the two isolates were analogous.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.     

条斑紫菜EST-SSR引物种间转移扩增真实性研究

当条斑紫菜EST-SSR引物在坛紫菜几个品系上进行种间扩增时,微卫星引物的扩增结果中也出现了多带形式。为了研究的假阳性问题,扩增产物被从胶上回收克隆测序。在回收的坛紫菜扩增产物中,同阳性对照条斑紫菜扩增产物大小相同的产物中有微卫星序列重复,但在大分子量的条带中却没有,显现出典型的假阳性问题。在坛紫菜扩增中产生的假阳性带不适合进行建立在微卫星突变基础上的遗传研究,然而,在确定相关的遗传性状后,这些引物也可以用来进行品系鉴定等遗传研究。     

Do cyanobacteria enhance germination and growth of rice?

A screening of 133 cyanobacterial strains in logarithmic growth phase was done to study their effects on rice germination and growth. In unialgal, non axenic culture 30% of the strains had no effect, while 70% of the strains had a negative effect on germination. In contrast, growth of rice was stimulated by 21% of the isolates and inhibited by 12%. Although 57% of the unicellular strains had a positive effect and many Nostoc strains had a negative one, it was not possible to correlate specific effects with taxonomic groups. Among the eight strains showing a stimulatory effect on growth only Anabaena 77S19 remained effective in axenic culture. Partitioning Anabaena 77S19 exudates into three fractions revealed that the organic fraction was more inhibitory. From this work it is concluded that presoaking rice seeds in a cyanobacterial culture should be done with caution or avoided altogether.     

条斑紫菜粗多糖对淋巴细胞和支持细胞的增殖作用

目的:探讨条斑紫菜粗多糖的一些生物学功能,包括促进淋巴细胞增殖以及促进支持细胞生长的功能。方法:将原代培养的大鼠和小鼠脾淋巴细胞以及大鼠睾丸支持细胞分别接种于96孔板中,将不同浓度的条斑紫菜粗多糖(0.05、0.5、5、10mg/mL)加入培养,每种浓度设6个复孔,并设对照组孔。采用四氮甲唑蓝(MTT)法检测细胞的增殖率。结果:条斑紫菜粗多糖能够显著地促进大鼠、小鼠脾淋巴细胞和大鼠睾丸支持细胞的增殖,增殖率随多糖浓度的提高而升高;当多糖浓度为10mg/mL时,增殖率分别为137.3%、149%和454.5%。结论:条斑紫菜粗多糖具有提高免疫力及促进生殖功能的作用,其对睾丸支持细胞的增殖作用目前还未见报道。     

几种藻类蛋白核的超微结构研究

应用电镜及免疫电镜技术对莱茵藻、小球藻、条浒苔和紫菜等藻类的叶绿体蛋白核的超微结构及主要组成成分进行了观察和研究。结果显示：不同藻类的蛋白核结构不同,显示了藻类蛋白核的多样性。蛋白核为球形或椭圆形,由蛋白质组成。莱茵藻、小球藻和条浒苔的蛋白核外围被淀粉鞘所包围,而紫菜的蛋白核外围无淀粉鞘而直接被叶绿体的类囊体所包围。淀粉鞘由淀粉组成,淀粉鞘的厚薄与藻体藻龄及增养状态有关系。在蛋白核中央,一般都具有由类囊体形成的孔道,使蛋白核与外界联系,小球藻和条浒苔蛋白核具有1条纵向孔道,而莱茵藻和紫菜为多条孔道。金相免疫技术检测结果显示Rubisco和Rubisco活化酶均在蛋白核及淀粉鞘区域中定位,表明蛋白核具有光合作用功能．     

LIFE HISTORY OF COLPOMENIA SINUOSA (SYCTOSIPHONACEAE,PHAEOPHYCEAE) IN THE AZORES1

Colpomenia sinuosa (Mertens ex Roth) Derbès and Solier (Scytosiphonaceae, Phaeophyceae) is a common species on the rocky intertidal shores of the Azores, where reproductive gametophytes occur throughout the year. Life‐history studies of this species were carried out in culture, and both sexual and asexual reproduction were observed. Anisogamous gametes fused to form zygotes. The zygotes gave rise to a filamentous prostrate sporophyte generation bearing unilocular sporangia, under both short‐day and long‐day conditions at 15 and 22°C, and to both unilocular and plurilocular sporangia, under the lower temperature condition. Unispores developed into gametophytes, and plurispores gave rise to filamentous sporophytes. Asexual reproduction was carried out by unfused female gametes and asexual plurispores produced from the same gametophyte. Unfused gametes developed into filamentous prostrate sporophytes producing unilocular sporangia in both culture conditions, and unispores released from the sporangia gave rise to gametophytes. Asexual plurispores from field gametophytes, under both culture conditions, developed directly into new gametophytes. The species exhibited three types of life history: a heteromorphic, diplohaplontic; a heteromorphic, monophasic (both with alternation between the erect and filamentous prostrate thalli); and a monomorphic, monophasic.