Cultivation of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura: mass production of zygote-derived seedlings under commercial cultivation conditions, a case study experience

Mariculture of the brown alga Hizikia fusiformis (Harvey) Okamura as an export-oriented human food has been there more for than 20 years in China. It is now one of the five major farmed algal species along the Chinese coast. Stable and sufficient supply of young seedlings for scaling up the cultivation has been a problem throughout the farming history of this species due to the unique dioecious life cycle and relatively short time window of sexual reproduction in nature. These two factors led to a practical difficulty in obtaining zygotes at identical developmental stage in viable amounts for seedling production. A key solution to this problem is to control the synchronization of the receptacle development and to realize the simultaneous discharge of male and female gametes, such that the fertilization rate could be greatly enhanced. Focusing on one of the farmed populations in this report, we present our results on mass production of seedlings using the synchronization technique on a large scale performed in 2007. Totally 5.5 hundred million embryos were obtained from 100 kg female sporophytes. The seedlings were raised up to 3.5 mm in length in greenhouse tanks over a month and were further grown in open sea for over 3 months at two experimental sites. The success of mass production of seedlings in this alga helped to lay the basis for future trials in other species in the genus of Sargassum that have identical life cycle.

Sex-linked microsatellite marker detected in the female gametophytes of Undaria pinnatifida (Phaeophyta)

P>In our microsatellite analysis of three male and three female gametophytes of Undaria pinnatifida (Harv.) Suringar, a microsatellite marker (part of the locus Up-AC-2A8, GenBank accession no. AY738602.1) was only polymerase chain reaction-amplified in three female gametophytes. This putative female-specific marker was further tested by the use of 32 male and 21 female gametophytes maintained in the Marine Biological Culture Collection Centre, China. In addition, three sporophytes were included for confirmation. Results showed that the marker was present in all of the female gametophytes and sporophyte cultures, but absent in all of the male gametophytes. To our knowledge, this is the first sex-related marker ever reported in U. pinnatifida. The discovery of this marker will accelerate gender identification and shed light on our understanding of the mechanisms of sex determination at a molecular level in this commercially important seaweed.

Variations in morphology and PSII photosynthetic capabilities during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta)

Background: Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae. Results: In the tetraspores, we observed two types of division, cruciate and zonate, and both could develop into multicellular bodies (disks). During the first 84 hours, tetraspores divided several times, but the diameter of the disks changed very little; thereafter, the diameter increased significantly. Scanning electron microscopy observations and analysis of histological sections revealed that the natural shape of the disk remains tapered over time, and the erect frond grows from the central protrusion of the disk. Cultivation of tissue from excised disks demonstrated that the central protrusion of the disk is essential for initiation of the erect frond. Photosynthetic (i.e., PSII) activities were measured using chlorophyll fluorescence analysis. The results indicated that freshly released tetraspores retained limited PSII photosynthetic capabilities; when the tetraspores attached to a substrate, those capabilities increased significantly. In the disk, the PSII activity of both marginal and central cells was similar, although some degree of morphological polarity was present; the PSII photosynthetic capabilities in young germling exhibited an apico-basal gradient. Conclusions: Attachment of tetraspores to a substrate significantly enhanced their PSII photosynthetic capabilities, and triggered further development. The central protrusion of the disk is the growth point, may have transfer of nutritive material with the marginal cells. Within the young germling, the hetero-distribution of PSII photosynthetic capabilities might be due to the differences in cell functions.

Cultivation of the brown alga Sargassum horneri: sexual reproduction and seedling production in tank culture under reduced solar irradiance in ambient temperature

As a large conspicuous intertidal brown alga, individuals of Sargassum horneri can reach a length of more than 7 m with a fresh weight of 3 kg along the coasts of the Eastern China Sea. The biomass of this alga as a vital component in coastal water ecology has been well documented. In recent years, a steady disappearance of the algal biomass along the once densely populated coastal areas of the Eastern China Sea has drawn attention in China. Efforts have been made to reconstruct the subtidal algal flora or even to grow the alga by use of long-lines. As part of the efforts to establish an efficient technique for producing seedlings of S. horneri, in this investigation a series of culture experiments were carried out in indoor raceway and rectangular tanks under reduced solar irradiance at ambient temperature in 2007-2008. The investigation demonstrated that: (1) sexual reproduction of S. horneri could be accelerated in elevated temperature and light climates, at least 3 months earlier than in the wild; (2) eggs of S. horneri had the potential to be fertilized up to 48 h, much longer than that of known related species; (3) suspension and fixed culture methods were both effective in growing the seedlings to the long-line cultivation stage; and (4) the life cycle of S. horneri in culture could be shortened to 4.5 months, thus establishing this alga as an appropriate model for investigating sexual reproduction in dieocious species of this genus.

Suspension culture of gametophytes of transgenic kelp in a photobioreactor

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l(-1) was obtained in batch culture with an initial dry cell weight of 126 mg l(-1) and with aeration rate of 1.2 l air min(-1) l(-1) culture, nitrate at 1.5 mM and phosphate at 0.17 mM. The yield of rtPA was 56 mu g g(-1) dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.

Transforming kelp into a marine bioreactor

The past decade has seen the genetic engineering of various types of seaweed. To date, genetic transformation studies have been carried out in several seaweeds, including the red seaweeds Porphyra, Gracilaria, Grateloupia, Kappaphyclus and Ceramium and the green seaweed Ulva. A genetic transformation model system has been established in the most commonly cultivated seaweed, the brown seaweed Laminaria japonica (kelp), based on the transfer of technology used in land plant transformation and also by modulating the seaweed life cycle. This model showed the potential for application of transgenic kelp to the production of valuable products and an indoor cultivation system for transgenic kelp was proposed, taking into account necessary factors for bio-safety. In this review, the establishment at use of the kelp transformation model is introduced, highlighting the potential for transforming kelp into a marine bioreactor.

Breaking seasonal limitation: year-round sporogenesis in the brown alga Laminaria saccharina by blocking the transport of putative sporulation inhibitors

Year-round induction of sporogenesis of Laminaria saccharina was performed by mechanically blocking the transport of the putative sporulation inhibitors produced by the blade meristem and culturing the plants in constant short days. Sporogenesis was successfully induced by removal of the blade meristem, either by cultivating distal blade fragments or by performing a transverse cut in the frond. The earliest sorus formation after artificial induction was 10 days. The age of the sporophytes used for induction was 6-11 months or 2 years in tank-grown or field-collected sporophytes, respectively. Zoospores were successfully released in all cases. Thus, by year-round artificial induction of sporogenesis, (1) sporeling production of L. saccharina and thereafter sporophyte cultivation could be achieved without seasonal limitation, and (2) the life cycle of L. saccharina (from spore to spore) could be completed within 8 months under controlled conditions. (C) 2004 Elsevier B.V. All rights reserved.

Mass culture of Undaria gametophyte clones and their use in sporeling culture

Undaria pinnatifida (Harv.) Sur. is one of the three main seaweed species under commercial cultivation in China. In the mid-1990s the annual production was about 20000 tons dry. The supply of healthy sporelings is key to the success of commercial cultivation of Undaria. Previous studies demonstrated that instead of the zoospore collection method, sporelings can be cultured through the use of gametophyte clones. This paper reports the experimental results on mass culture of clones and sporeling raising in commercial scale. Light had an obvious effect on growth of gametophyte clones. Under an irradiance of 80 mumol m(-2) s(-1) and favorable temperature of 22-25degreesC, mean daily growth rate may reach as high as 37%. Several celled gametophyte fragments were sprayed onto the palm rope frame. Gametogenesis occurred after 4-6 days. Juvenile sporeling growth experiments showed that nitrate and phosphate concentrations of 2.9 10(-4) mol 1(-1) and 1.7 10(-5) mol 1(-1) were sufficient to enable the sporelings to maintain a high daily growth rate. Sporelings can reach a length of 1 cm in a month. Since 1997, extension of the clone technique has been carried out in Shandong Province. Large-scale production of sporelings for commercial cultivation of 14 and 31 hectares in 1997 and 1998 had been conducted successfully.

Three-stage transformation of chlorophyll transient fluorescence pattern under sustained dehydration and the discovery of critical water content in seaweeds

The chlorophyll fluorescence kinetics of marine red alga Grateloupia turutunt Yamada, green alga Ulva pertusa Kjellm and brown alga Laminaria japonica Aresch during natural sustained dehydration were monitored and investigated. The pulse amplified modulation (PAM) system was used to analyze the distinct fluorescence parameters during thallus dehydration. Results proved that the fluorescence kinetics of different seaweed all showed three patterns of transformation with sustained water loss. These were: 1) peak kinetic pattern (at the early stage of dehydration fluorescence enhanced and quenched subsequently, representing a normal physiological state). 2) plateau kinetic pattern (with sustained water loss fluorescence enhanced continuously but quenching became slower, finally reaching its maximum). 3) Platform kinetic pattern (fluorescence fell and the shape of kinetic curve was similar to plateau kinetic pattern). A critical water content (CWC) could be found and defined as the percentage of water content just prior to the fluorescence drop and to be a significant physiological index for evaluation of plant drought tolerance. Once thallus water content became lower than this value the normal peak pattern can not be recovered even through rehydration, indicating an irreversible damage to the thylakoid membrane. The CWC value corresponding to different marine species were varied and negatively correlated with their desiccation tolerance, for example. Laminaria japonica had the highest CWC value (around 90%) and the lowest dehydration tolerance of the three. In addition, a fluorescence "burst" was found only in red algae during rehydration. The different fluorescence parameters F-o, F-v and F-v, F-m were measured and compared during water loss. Both F-o and F-v increased in the first stage of dehydration but F-v/F-m. kept almost constant. So the immediate response of in vivo chlorophyll fluorescence to dehydration was an enhancement. Later with sustained dehydration F-o increased continuously while F-v decreased and tended to become smaller and smaller. The major changes in fluorescence (including fluorescence drop during dehydration and the burst during rehydration) were all attributed to the change in F-o instead of F-v This significance of F-o indicates that it is necessary to do more research on F-o as well as on its relationship with the state of thylakoid membrane.

Study on early-stage development of conchospore in Porphyra yezoensis Ueda

Porphyra yezoensis Ueda (Rhodophyta) is a seaweed of economic importance with a typical dimorphic life cycle consisting of a leafy gametophyte and a filamentous sporophyte. Recently, it has been recognized as a model system for fundamental and applied studies in marine biological sciences. Conchospore, a major spore linking the two distinct multicellular phases in the life cycle, is most widely used in the breeding of P. yezoensis. In this paper, the early-stage development of conchospore, including the attachment and the cell wall formation, was studied with fluorescent reagents staining and Scanning Electron Microscopy detection. Results displayed: (I) the cell wall began to be generated after culturing for 4 h in the attached conchospores; (2) the initially released conchospores were plastids with some filmy, amorphous substance on the surface, and they attached to the fibers firmly via the actively secreted mucilaginous substances after their touch to the fibers; (3) cellulase and pectolase prohibited the attachment of conchospores in the different ways; and (4) only attached conchospores generated cell walls and developed normally, while the suspending ones could not. It indicated that the cellulose played crucial roles in the permanent attachment as the pectin did in the initial attachment. The conchospore attachment seemed to trigger the cell wall formation and the further development. Affects of light on the development of conchospores were also discussed. The results showed that high intensity (200 mu mol.m(-2).s(-1)) and long-wave (>= 580 nm) light facilitated the division rate of conchospores. (C) 2008 Elsevier B.V. All rights reserved.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: stress resistance of artificially raised young seedlings revealed by chlorophyll fluorescence measurement

Commercial farming of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura in China and South Korea in the sea depends on three sources of seedlings: holdfast-derived regenerated seedlings, young plants from wild population and zygote-derived seedlings. Like many successfully farmed seaweed species, the sustainable development of Hizikia farming will rely on a stable supply of artificial seedlings via sexual reproduction under controlled conditions. However, the high rate of detachment of seedlings after transfer to open sea is one of the main obstacles, and has limited large-scale application of zygote-derived seedlings. To seek the optimal condition for growing seedlings on substratum in land-based tanks for avoidance of detachment in this investigation, young seedlings were grown in both outdoor tanks exposed directly to sunlight and in indoor raceway tanks in reduced, filtered sunlight. Results showed that young seedlings, immediately after fertilization, could withstand a daily fluctuation of direct solar irradiance up to a level of 1800 mu mol photons m(-1)s(-1), and maintained a faster growth rate than seedlings grown in indoor tanks. Detailed experiments by use of chlorophyll fluorescence measurements further demonstrated that the overnight (12 h) recovery of optimal fluorescence quantum yield (F-v/F-m) of seedlings after 1 h treatment at 40 degrees C was 98%, and the 48 h recovery of F-v/F-m of seedlings after 1 h exposure to 1800 mu mol m(-2)s(-1) was 92%. Forty-one-day-old seedlings showed no significant decrease of optimal fluorescence quantum yield at salinity ranging from 30 to 5 ppt for a treatment up to 17 h. Six-hour desiccation treatment did not have any influence on the optimal fluorescence quantum yield. Exposure to 18 mmol L-1 sodium hypochlorite for 10 min did not damage the PSII efficiency, and thus could be used to remove epiphytic algae. The strong tolerance of young seedlings to high temperature, high irradiance, low salinity and desiccation found in this investigation supports the view that mass production of Hizikia seedlings should be performed in ambient light and temperature instead of in shaded greenhouse tanks.

Temperature and light tolerance of representative brown, green and red algae in tumble culture revealed by chlorophyll fluorescence measurements

Laminaria japonica, Undaria pinnatifida, Ulva lactuca, Grateloupia turuturu and Palmaria palmata are Suitable species that fit the requirements of a seaweed-animal integrated aquaculture system in terms of their viable biomass, rapid growth and promising nutrient uptake rates. fit this investigation, the responses of the optimal chlorophyll fluorescence yield of the five algal species in tumble Culture were assessed at a temperature range of 10 similar to 30 degrees C. The results revealed that Ulva lactuca was the most resistant species to high temperature, withstanding 30 degrees C for 4 h without apparent decline in the optimal chlorophyll fluorescence yield. While the arctic alga Palmaria palmata was the most vulnerable one, showing significant decline in the optimal chlorophyll fluorescence yield at 25 degrees C for 2 h. The cold-water species Laminaria japonica, however, demonstrated strong ability to cope with higher temperature (24 similar to 26 degrees C) for shorter time (within 24 h) without significant decline in the optimal chlorophyll fluorescence yield. Grateloupia turuturu showed a general decrease in the optimal chlorophyll fluorescence yield with the rising temperature from 23 to 30 degrees C, similar to the temperate kelp Undaria pinnatifida. Changes of chlorophyll fluorescence yields of these algae were characterized differently indicating the existence of species-unique strategy to cope with high light. Measurements of the optimal chlorophyll fluorescence yield after short exposure to direct solar irradiance revealed how long these exposures could be without significant photoinhibition or with promising recovery in photosynthetic activities. Seasonal pattern of alternation of algal species in tank culture in the Northern Hemisphere at the latitude of 36 degrees N was proposed according to these basic measurements.

Temperature tolerance of young sporophytes from two populations of Laminaria japonica revealed by chlorophyll fluorescence measurements and short-term growth and survival performances in tank culture

The cold-water subtidal brown alga Laminaria japonica has been commercially fanned in the Far East and has been on top of all marine-fanned species in terms of farming area and annual output worldwide. The successful trials of transplantation of young sporophytes from the north to the south in winter along the Chinese coast in the 1950s led to the spreading of cultivation activities down to a latitude of 25-26 degrees N. Up to today, nearly 50% of the annual output of this farmed alga, as a cold-water species, comes from the sub-tropical south in China. The demand to have high-temperature-tolerant strains/ecotypes in farming area calls for a practical method to judge and select the desired parental plants for breeding programs and for seedling production. In this paper, we report our results on using chlorophyll fluorescence measurement and short-term growth performance in tank culture to estimate the temperature tolerance of offspring from two populations, Fujian Farmed Population (FFP) sampled from Fujian province (latitude: 25-26 degrees N) in subtropical area and Qingdao Wild Population (QWP) sampled from Qingdao (latitude: 36 degrees N). Contrary to what has been usually thought, the results revealed that offspring from Qingdao wild population in the north showed better performance both in short-term growth and survival rates and in optimal quantum efficiency (F-v/F-m) when exposed to higher temperature (20-25 degrees C). This result was further confirmed by fluorescence quenching analysis. QWP distributed along the southern distribution limit at a latitude of 36 degrees N in the Pacific west coast is thus taken as a more ideal one than the fanned population in subtropical region as a source of parental plants for breeding high-temperature-tolerant varieties. (c) 2006 Elsevier B.V. All rights reserved.

Circadian rhythms in the growth and reproduction of the brown alga Undaria pinnatifida and gametogenesis under different photoperiods

Circadian growth rhythm of the juvenile sporophyte of the brown alga Undaria pirznatifida was measured with the computer-aided image analysis system in constant florescent white light under constant temperature (10 degrees C). The growth rhythm persisted for 4 d in constant light with a free-running period of 25. 6 h. Egg release from filamentous gametophytes pre-cultured in the light - dark regime was evaluated for six consecutive days at fixed time intervals in constant white light and 12 h light per day. Egg release rhythm persisted for 3 d in both regimes, indicating the endogenous nature. Temporal scale of egg release and gametogenesis in 18, 16, 12 and 8 h light per day were evaluated respectively using vegetatively propagated filamentous gametophytes. Egg release occurred 2 h after the onset of dark phase and peaked at midnight. Evaluation of the rates of oogonium formation, egg release or fertilization revealed no significant differences in four light-dark regimes, indicating; the great plasticity of sexual reproduction. No photoperiodic effect in gametogenesis in terms of oogonium formation and egg release was found, but fertilization in short days was significantly higher than in long days. Results of this investigation further confirmed the general occurrence of circadian rhythms in inter-tidal seaweed species.

Evaluation of 28 marine algae from the Qingdao coast for antioxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae)

The extracts obtained from 28 species of marine algae were evaluated for their antioxidant activity (AA) versus the positive controls butylated hydroxytoluene (BHT), gallic acid (GA), and ascorbic acid (AscA). Most of the tested samples displayed antioxidant activity to various degrees. Among them, the extract of Symphyocladia latiuscula exhibited the strongest AA, which was comparable to BHT, GA, and AscA in radical scavenging activity, as shown in the DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) assay, and higher than those of the positive controls in beta-carotene-linoleate assay system. In addition, the ethyl acetate-soluble fraction isolated from the crude extract of S. latiuscula exhibited the highest antioxidant activity in both assay systems. This fraction was further fractionated into seven subfractions (F1-F7) by vacuum liquid chromatography (VLC). F1 and F4 were found to be the most effective subfractions in scavenging DPPH radical assay and in the beta-carotene-linoleate assay, respectively. The total phenolic content (TPC) and reducing power (RP) for all of the extracts, fractions, and subfractions (F1-F7) were also determined. The TPC of the 28 extracts ranged from 0.10 to 8.00 gallic acid equivalents (mg/g seaweed dry weight) while the RP ranged from 0.07 to 11.60 ascorbic acid equivalents (mg center dot g(-1) seaweed dry weight). Highly positive relationships between AA and TPC as well as between AA and RP were found for the extracts and fractions, while for the subfractions F1-F7 only weak or no such relations were found. The results obtained from this study indicate that further analysis is needed of those marine algal species that contain the most antioxidant activity in order to identify the active principles.