Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

营养盐因子对细基江蓠繁枝变种氮、磷吸收速率的影响

在实验室条件下,研究了营养盐因子对细基江蓠繁枝变种的氮、磷吸收速率的影响。分别进行了营养盐浓度与温度双因子试验,氮磷比与荧 浓度双因子试验肽不同化合态氮比例单因子试验。（1）氮、磷的吸收速率随营养盐浓度的升高而增大,氮的吸收速率在21℃,总氮浓度为100μmol/L时最大,达2.58μmol/(g.h);磷的吸收速率在31℃,总磷为6．3μmol/L时最大,达0.17μmol/(g.h),温度与营养盐浓度有显著的交互作用效应。（2）当氮浓度一定时,环境氮磷比对氮的吸收率无显著影响,但对磷的吸收速率有显著影响,藻中收的氮磷比随环境氮磷比的不同而变化,（3）对3种不同化合态氮的吸收速率与培养液中各种氮占总无机氮的比例呈正相关,当三比例相同时,对NH4^ -N、NO3^--N格NO2^-析吸收分别占总吸收氮的40．7％、28．5％和30．8％。     

Purification of plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata

We report a straightforward protocol for isolating plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata. Plastids were purified using differential centrifugation and 2-step sucrose density gradients. We found that 10% polyethylene glycol 4000 was essential for maintaining plastid integrity prior to lysis. Plastid DNA isolated directly from the purified rhodoplasts was sufficiently pure for restriction endonuclease fragment analyses. Database comparisons of sequences generated randomly from a shotgun genomic library indicated that plastid DNA was 89% pure following ultracentrifugation in isopycnic cesium chloride equilibrium gradients. The protocol yields 30–50 μg of plastid DNA per 100 g of fresh algal tissue.     

Effects of plant growth regulators on callus formation, growth and regeneration in axenic tissue cultures of Gracilaria tenuistipitata and Gracilaria perplexa (Gracilariales, Rhodophyta)

The effects of auxins and cytokinin on callus formation, growth and regeneration of Gracilaria tenuistipitata Chang et Xia and G. perplexa Byrne et Zuccarello (Gracilariales, Rhodophyta) are reported. Plant growth regulators (PGR) in concentrations ranging from 0.1 to 100.0 μmol of indole‐3‐acetic acid, 2,4‐dichlorophenoxyacetic acid (2,4‐D), and kinetin (K) were added to the ASP 12‐NTA solid medium (0.7% agar), and apical and intercalary segments (5 mm long) were inoculated as initial explants. K stimulated growth rates of intercalary segments of G. tenuistipitata in a linear relation, and 2,4‐D (1.0 μmol) and K (10.0 μmol) stimulated growth rates of apical and intercalary segments of G. perplexa, respectively. The simultaneous formation of apical, basal, and intermediate calluses is reported for the first time in axenic tissue cultures of red algae. With intercalary segments of G. tenuistipitata, basal callus induction rates were higher than those of apical and intermediate calluses in the majority of treatments, and auxins had stimulatory effects on the formation of all callus types. In apical segments of G. perplexa, intermediate callus formation was stimulated only by treatment with 1.0 μmol of K, while apical callus formation was stimulated by indole‐3‐acetic acid (1.0–10.0 μmol), 2,4‐D (10.0–100.0 μmol), or K (0.1 μmol). Intercalary segments of G. perplexa developed only intermediate calluses, and the majority of treatments with PGR stimulated higher rates than those presented by apical segments. Potential for regeneration (development of adventitious plantlets originated from callus cells) was higher in apical calluses than in basal and intermediate calluses developed in intercalary segments of G. tenuistipitata. Moreover, auxins and cytokinin were essential to the induction of regeneration in intermediate calluses, while specific concentrations stimulated regeneration from basal and apical calluses. Plant regeneration in G. perplexa was observed only after transferring calluses from solid to liquid medium, and the majority of treatments with PGR had stimulatory effects. Regenerating plants of G. perplexa developed tetrasporangia, and released tetraspores giving rise to adult gametophytes. Our results indicate that auxins and cytokinin have a regulatory role in the growth and morphogenesis in G. tenuistipitata and G. perplexa, and diversity of responses presented by both species is related to specific developmental systems.     

Testing the effects of heterozygosity on growth rate plasticity in the seaweed Gracilaria chilensis (Rhodophyta)

Heterozygosity has been positively associated with fitness and population survival. However, the relationship between heterozygosity and adaptive phenotypic plasticity (i.e., plasticity which results in fitness homeostasis or improvement in changing environments) is unclear and has been poorly explored in seaweeds. In this study, we explored this relationship in the clonal red seaweed, Gracilaria chilensis by conducting three growth rate plasticity experiments under contrasting salinity conditions and by measuring heterozygosity with five microsatellite DNA markers. Firstly, we compared growth rate plasticity between the haploid and diploid phases. Secondly, we compared growth rate plasticity between diploids with different numbers of heterozygous loci. Finally, we compared growth rate plasticity between diploid plants from two populations that are expected to exhibit significant differences in heterozygosity. We found that, (i) diploids displayed a higher growth rate and lower growth rate plasticity than haploids, (ii) diploids with a higher number of heterozygous loci displayed lower growth rate plasticity than those exhibiting less heterozygosity, and (iii) diploid sporophytes from the population with higher heterozygosity displayed lower growth rate plasticity than those with lower heterozygosity. Accordingly, this study suggests that heterozygosity is inversely related to growth rate plasticity in G. chilensis. However, better genetic tools in seaweeds are required for a more definitive conclusion on the relationship between heterozygosity and phenotypic plasticity.     

Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content

Select species of the agarophyte Gracilaria were studied from southeastern Australia. The morphology and anatomy of species is described and molecular relations are inferred based on plastid and mitochon‐drial DNA sequence data. Agar yields and qualities are determined for each species. Gracilaria chilensis, found in Tasmania and Victoria, is morphologically and molecularly similar to G. chilensis from New Zealand and Chile and has low agar yields of 11–16%. Gracilaria cliftonii from Victoria, has high crude agar yield (52%) and is molecularly uniform. Gracilaria perplexa sp. nov., known only from Botany Bay, New South Wales, has an agar yield of 39%. The agar of G. perplexa is unusual in requiring the addition of 0.1 mol L^?1 NaCl for alcohol precipitation and is cold‐water (25°C) soluble because of the very high sulfate ester content. Molecular phylogeny shows that G. perplexa is closely related to Gracilaria preissiana from western Australia, but differs from the latter in its reduced branching and narrower more terete axes.     

STUDIES OF MESOHERBIVORY IN AQUARIA AND IN AN UNBARRICADED MARICULTURE FARM ON THE CHINESE COAST1

The effects upon macrophytic and epiphytic algae of caprellid amphipods, including Caprella irregularis Mayer, and of gammarid amphipods, including Ampithoe lacertosa Bate, Jassa falcata (Montagu) and Pontogeneia rostrata Gurjanova, were studied in aquaria and in a coastal mariculture farm near Qingdao, People's Republic of China. The macrophytic species studied included Enteromorpha linza (L.) J. Agardh, Gracilaria asiatica Zhang & Xia, Gracilaria lemaneiformis (Bory) Weber-van Bosse and Scytosiphon lomentaria (Lyngbye) C. Agardh. These algae and amphipods were selected for study because of their local abundances. Aquarium experiments demonstrated that C. irregularis, J. falcata and P. rostrata feed on ephemeral/epiphytic algae. A. lacertosa (adults and juveniles) ate Gracilaria but preferred E. linza. The different species of amphipods had substantially different patterns of daily activity. Caprellids prevented epiphytic overgrowth of G. asiatica when added to plants in an experimental, openocean, mariculture farm, despite the presence of numerous amphipod predators (e.g. the fishes Hexagrammos otakii Jordan & Starks and Sebastes thompsoni Jordan & Hubbs). Periodic removals of all (naturally colonizing) amphipods from another group of plants in the farm resulted in heavy epiphytic overgrowth. Since these results were obtained in an unbarricaded mariculture farm, we conclude that mesoherbivores may have large effects upon algal community structure in temperate intertidal and subtidal habitats.     

GENETICS OF GRACILARIA TIKVAHIAE (RHODOPHYCEAE). XI. FURTHER CHARACTERIZATION OF A BISEXUAL MUTANT1,2

A spontaneous bisexual mutant of Gracilaria tikvahiae McLachlan has been further characterized. Female plants that are carriers for the mutation, but do not themselves express bisexuality, have been identified among progeny derived from the original bisexual male plant. In crosses to normal males these carrier females yielded normal tetrasporophytes which in the subsequent gametophyte generation produced a 2 female: 1 male: 1 bisexual segregation. In crosses to bisexual males the carrier females produced unusual tetrasporophytes that formed cystocarps in addition to tetraspores. The gametophyte generation obtained from the tetraspores of these tetrasporophytes included only females and bisexuals, these being present in a 1:1 ratio. Other crosses, using bisexual male progeny, indicated that these have the same characteristics as the original bisexual mutant. All of the results are consistent with the genetic interpretation made previously that bisexuality results from a single recessive mutation, designated bi, in a gene distinct from the mt locus controlling male vs. female differentiation. From the phenotypes that have been observed it appears that the mutation does not cause bisexuality per se but rather results in unregulated female expression in males and tetrasporophytes where female-specific genes are normally repressed. It is suggested that the normal bi⁺ allele plays an important role in that repression process. The origin of the bisexual mutation has been re-examined by studying the progenitor stock of the mutation and stocks related to it. It appears that the bisexual mutation arose in a two-step process, first to a low level of expression that is found in the progenitor stocks and then to the high level of expression found in mutant clone 1045(bi) and its descendants.     

Mechanisms of inorganic carbon acquisition in Gracilaria gaditana nom. prov. (Rhodophyta)

The mechanisms for acquisition of dissolved inorganic carbon (DIC) in the red macroalga Gracilaria gaditana nom. prov. have been investigated. The capacity for HCO₃ ⁻ use by an extracellular carbonic anhydrase (CA; EC 4.2.1.1), and by an anion exchanger with similar properties to that of red blood cells (AE1), has been quantified. It was illustrated by comparing O₂ evolution rates with those theoretically supported by CO₂, as well as by photosynthesis-pH curves. Both external and internal CA, and a direct uptake were involved in HCO₃ ⁻ use, since photosynthesis and pH evolution were affected by acetazolamide, 6-ethoxyzolamide (inhibitors of external and total CA, respectively) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, (DIDS; an inhibitor of HCO₃ ⁻ exchanger protein). The activity of the external CA was detected by a potentiometric method and by an alternative method based on the study of O₂ evolution after addition of CO₂ and acetazolamide. The latter method showed a residual photosynthetic rate due to direct HCO₃ ⁻ use. Inhibitors caused a reduction in the pH compensation points in pH-drift experiments. The CO₂ compensation points for photosynthesis increased when the inhibitors were applied, indicating a suppresion of the pathways involved in the carbon-concentrating mechanism. The net photosynthesis rates as a function of DIC concentration displayed a biphasic pattern that could be supported by the occurrence of the two mechanisms of HCO₃ ⁻ use. The potential contribution to HCO₃ ⁻ acquisition by the DIDS-sensitive mechanism was higher after culturing at a high pH. Our results suggest that the HCO₃ ⁻ use by Gracilaria gaditana is carried out by the two DIC uptake mechanisms. These operate simultaneously with different affinities for DIC, the indirect HCO₃ ⁻ use by an external CA activity being the main pathway. The presence of a carbon-concentrating mechanism confers eco-physiological advantages in a fluctuating ecosystem subjected daily to high pHs and low DIC concentrations. Received: 3 July 1998 / Accepted: 30 November 1998     

Chemical characteristics and gelling properties of agar from two Philippine Gracilaria spp. (Gracilariales, Rhodophyta)

The chemical structure of agars extracted from Philippine Gracilaria arcuata and G. tenuistipitata were determined by NMR and infrared spectroscopy. Agar with alternating 3-linked 6-O-methyl-β-D-galactopyranosyl and 4-linked 3,6-anhydro-2- O-methyl-α-L-galactopyranosyl units was isolated from G. arcuata, while the agar from G. tenuistipitata possesses the regular agarobiose repeating unit with partial methylation at the 6-position of the D-galactosyl residues. Both agars exhibit sulphate substitution at varying positions in the polymer. Chemical analyses reveal higher 3,6-anhydrogalactose and lower sulphate contents in alkali-modified than in native agar from both samples. Also, alkali modification enhanced agar gel strength and syneresis. Native G. arcuata agar produces a viscous solution (2000 cP at 75 °C) with a high gelling point (>60 °C) that forms a soft gel even after alkali modification (gel strength: <300 g cm⁻²). On the other hand, the agar from G. tenuistipitata exhibits gel qualities typical of most Gracilaria agars. This revised version was published online in June 2006 with corrections to the Cover Date.